METHODS AND PRODUCTS RELATING TO GSK3ß REGULATION

ABSTRACT

The invention relates to methods and compositions for regulation of GSK3β activity. The invention provides phosphorylated GSK3β polypeptides and antibodies that recognize such polypeptides The invention further includes methods for treating disorders that are associated with elevated or reduced GSK3β activity.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/887,679, filed May 6, 2013, now pending, which is a continuation ofU.S. application Ser. No. 12/989,901, filed Jan. 31, 2011, now U.S. Pat.No. 8,445,648, issued on May 21, 2013, which is a national stage filingunder U.S.C. §371 of PCT International application PCT/US2009/002609,filed Apr. 29, 2009 which was published under PCT Article 21(2) inEnglish, which claims the benefit under 35 U.S.C. 119(e) of U.S.provisional application Ser. No. 61/126,133, filed Apr. 30, 2008, theentire content of each referenced application is incorporated byreference herein in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under NIH R01 AI051454awarded by the National Institutes of Health. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The invention relates to methods and compositions for regulating GSK3βactivity. The invention in some aspects includes phosphorylated GSK3βpolypeptides, and methods for using these polypeptides in treatment ofdiseases or conditions characterized by GSK3β activity.

BACKGROUND OF INVENTION

Glycogen Synthase Kinase 3β (GSK3β) is a serine/threonine kinase thatregulates many aspects of cell function such as gene expression,apoptosis and metabolism, through phosphorylation of a wide variety ofcellular substrates. One of the GSK3β substrates is the Wnt signalingmolecule β-catenin. Phosphorylation of β-catenin by GSK3β targetsβ-catenin for ubiquitination and subsequent degradation. Inhibition ofGSK3β activity through phosphorylation is the primary mechanism thatregulates this widely expressed active kinase. The protein kinase Aktinhibits GSK3β by phosphorylation at N-terminal serine residues.However, preventing Akt-mediated phosphorylation does not affect thecell survival pathway activated through the GSK3β substrate β-catenin.GSK3β activity is also enhanced by phosphorylation on several tyrosineresidues. In some instances GSK3β activity is regulated through itssub-cellular localization. The activity of GSK3β is linked to multipledisorders including neurological disorders, diabetes, and cancer.

SUMMARY OF INVENTION

Aspects of the invention described herein resale to the discovery thatp38 mitogen-activated protein kinase (MAPK) inactivates GSK3β by directphosphorylation at its C-terminus, and this inactivation can lead to anaccumulation of β-catenin. p38 MAPK-mediated phosphorylation of GSK3βoccurs at least in brain and thymocytes. Activation ofβ-catenin-mediated signaling through GSK3β inhibition may provide amechanism for p38 MAPK-mediated survival in specific tissues.

According to an aspect of the invention, isolated GSK3β polypeptide areprovided. The isolated polypeptides are fragments of a full-length GSK3βprotein and include a phosphorylated residue that corresponds to residueThr³⁹⁰ in a full-length, wild-type, human GSK3β amino acid sequence, orcorresponds to residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3βamino acid sequence. In some embodiments, the GSK3β polypeptide is ahuman GSK3β polypeptide. In certain embodiments, the phosphorylatedresidue corresponds to the Thr³⁹⁰ residue. In some embodiments, thefragment includes the amino acid sequence set forth as:RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7). In some embodiments, the GSK3βpolypeptide is a mouse GSK3β polypeptide. In certain embodiments, thephosphorylated residue corresponds to the Ser³⁸⁹ residue. In someembodiments, the fragment includes the amino acid sequence set forth asARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87).

According to another aspect of the invention, compositions are provided.The compositions include any isolated GSK3β polypeptide of anyembodiment of the aforementioned aspect of the invention and apharmaceutically acceptable carrier.

According to another aspect of the invention, fusion proteins areprovided. The fusion proteins include the GSK3β polypeptide of any oneof embodiment of the foregoing aspect of the invention.

According to yet another aspect of the invention, isolated antibodies orantigen-binding fragments thereof are provided. The isolated antibody orantigen-binding fragments thereof bind specifically to an epitope ofphosphorylated GSK3β polypeptide, wherein the epitope includes aphosphorylated residue that corresponds to residue Thr³⁹⁰ in afoil-length, wild-type, human GSK3β amino acid sequence, or correspondsto residue Ser³⁸⁹ in a full-lengths wild-type, mouse GSK3β amino acidsequence. In some embodiments, the full-length GSK3β protein is a humanGSK3β protein. In some embodiments, the phosphorylated residuecorresponds to the Thr³⁹⁰ residue. In certain embodiments, thefull-length GSK3β protein is a mouse GSK3β protein. In some,embodiments, the phosphorylated residue corresponds to the Ser³⁸⁹residue. In some embodiments, the antibody competitively inhibits thebinding of phospho-S³⁸⁹ GSK3β antibody to an epitope that includes aphosphorylated residue that corresponds to residue Ser³⁸⁹ in a fulllength mouse GSK3β protein, in some embodiments, the antibodyspecifically binds to the epitope with a binding affinity of about1×10⁻⁶ M, 1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 5×10⁻¹⁰ M, or 1×10⁻¹¹M or less. In certain embodiments, the antibody is phospho-S³⁸⁹ GSK3βantibody. In some embodiments, the antibody or antigen-binding fragmentthereof is attached to a detectable label.

According to an aspect of the invention, a nucleic acid is provided. Thenucleic acid encodes any antibody of an embodiment of the aforementionedaspect of the invention. According to another aspect of the invention, ahybridoma that includes the any of the aforementioned nucleic acidmolecules is provided. According to another aspect of the invention, ahybridoma cell line that produces the any of the aforementionedantibodies is provided. According to yet another aspect of theinvention, an expression vector that includes any of the aforementionedisolated nucleic acid molecules encoding the antibody or antigen-bindingfragment thereof are provided. According to another aspect of theinvention, a host cell is provided. The host cell is transformed by ortransacted with any of the aforementioned expression vectors. Accordingto another aspect of the invention, a plasmid that produces any of theaforementioned antibodies or antigen-binding fragments thereof isprovided.

According to yet another aspect of the invention, compositions thatinclude any of the aforementioned antibodies or antigen-bindingfragments thereof are provided. In some embodiments, the compositionsalso include a carrier. In certain embodiments, the carrier is apharmaceutically acceptable carrier.

According to yet another aspect of the invention, methods of making anantibody that specifically binds to phosphorylated GSK3β protein areprovided. The methods include immunizing an animal with the any of theaforementioned polypeptides of any of aspect of the invention. In someembodiments, the method also includes removing a lymph node from theimmunized animal, harvesting cells from the removed lymph node, fusingthe harvested cells with myeloma cells to make hybridomas, expanding thehybridomas, identifying a hybridoma that produces an antibody thatspecifically binds to the phosphorylated polypeptide, and collecting theantibody produced by the hybridoma. In certain embodiments, the methodalso includes harvesting immune cells from the immunized animal,isolating the antibody that specifically binds phosphorylated GSK3βprotein, sequencing the antibody, preparing a cell that expresses theantibody sequence, and collecting the expressed antibody. In someembodiments, the polypeptide that includes the amino acid sequence setforth as RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7). In some embodiments, thepolypeptide includes the amino acid sequence set forth asARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87).

According to yet another aspect of the invention, methods of producingan antibody that specifically binds to a phosphorylated GSK3βpolypeptide are provided. The methods include inoculating an animal withany polypeptide of any of the aforementioned aspects of the invention,in which the polypeptide includes a phosphorylated residue thatcorresponds to residue Thr³⁹⁰ in a full-length, wild-type, human GSK3βamino acid sequence, or corresponds to residue Ser³⁸⁹ in a full-length,wild-type, mouse GSK3β amino acid sequence, wherein the polypeptideelicits an immune response in the animal to produce the antibody; andisolating the antibody from the animal; wherein the antibodyspecifically binds to a phosphorylated GSK3β polypeptide. In certainembodiments, the polypeptide includes the amino acid sequence set forthas RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7). In some embodiments, thepolypeptide includes a threonine residue that corresponds to residueThr³⁹⁰ of full-length, wild-type, human GSK3β polypeptide. In someembodiments, the polypeptide includes the amino acid sequence set forthas ARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87). In certain embodiments, thepolypeptide includes a serine residue that corresponds to residue Ser³⁸⁹of full-length, wild-type, mouse GSK3β polypeptide.

According to yet another aspect of the invention, methods of reducingactivity of GSK3β in a cell are provided. The methods include contactingthe cell with a composition comprising an isolated GSK3β polypeptide,wherein the polypeptide is a full-length GSK3β protein or a fragment ofa full-length GSK3β protein and includes a phosphorylated residue thatcorresponds to residue Thr³⁹⁰ in a full-length, wild-type, human GSK3βamino acid sequence, or corresponds to residue Ser³⁸⁹ in a full-length,wild-type, mouse GSK3β amino acid sequence. In some embodiments, theGSK3β polypeptide is a human GSK3β polypeptide. In some embodiments, thephosphorylated residue corresponds to the Thr³⁹⁰ residue. In certainembodiments, the fragment includes the amino acid sequence set forth as;RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7). In some embodiments, the GSK3βpolypeptide is a mouse GSK3β polypeptide. In some embodiments, thephosphorylated residue corresponds to the Ser³⁸⁹ residue. In certainembodiments, the fragment includes the amino acid sequence set forth asARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87). In some embodiments, the methodsalso include contacting the cell with a compound and/or applying aprocedure to the cells that results in the phosphorylation of a GSK3βresidue that corresponds to residue Thr³⁹⁰ in a full-length, wild-type,human GSK3β amino acid sequence, or corresponds to residue Ser³⁸⁹ in afull-length, wild-type, mouse GSK3β amino acid sequence. In someembodiments, the compound or procedure modulates expression or activityof a component of the p38 MAPK signaling pathway in the cell. In certainembodiments, the compound or procedure increases expression or activityof p38 MAPK. In some embodiments, the compound includes p38 MAPK. Insome embodiments, the method is a method for treating a neurologicaldisease or condition.

According to yet another aspect of the invention, methods of reducingactivity of GSK3β in a cell are provided. The methods include contactingthe cell with a compound and/or applying a procedure to the cells thatresults in the phosphorylation of a GSK3β residue that corresponds toresidue Thr³⁹⁰ in a full-length, wild-type, human GSK3β amino acidsequence, or corresponds to residue Ser³⁸⁹ in a full-length, wild-type,mouse GSK3β amino acid sequence. In certain embodiments, the compound orprocedure modulates expression or activity of a component of the p38MAPK signaling pathway in the cell. In some embodiments, the compound orprocedure increases expression or activity of p38 MAPK. In someembodiments, the compound includes p38 MAPK. In certain embodiments, themethod is a method for treating a neurological disease or condition.

According to yet another aspect of the invention, methods for treating adisease or condition associated with GSK3β activity are provided. Themethods include administering to a subject having a disease or conditionassociated with GSK3β activity a therapeutically effective amount of acomposition that includes an isolated GSK3β polypeptide, wherein thepolypeptide is a full-length GSK3β protein or a fragment of afull-length GSK3β protein and includes a phosphorylated residue thatcorresponds to residue Thr³⁹⁰ in a full-length, wild-type, human GSK3βamino acid sequence, or corresponds to residue Ser³⁸⁹ in a full-length,wild-type, mouse GSK3β amino acid sequence, in some embodiments, theGSK3β polypeptide is a human GSK3β polypeptide. In some embodiments, thephosphorylated residue corresponds to the Thr³⁹⁰ residue. In certainembodiments, the polypeptide includes the amino acid sequence set forthas: RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7). In some embodiments, the GSK3βpolypeptide is a mouse GSK3β polypeptide. In some embodiments, thephosphorylated residue corresponds to the Ser³⁸⁹ residue. In certainembodiments, the fragment includes the amino acid sequence set forth asARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87). In some embodiments, the methodalso includes contacting the cell with a compound and/or applying aprocedure to the cells that results in the phosphorylation of a GSK3βresidue that corresponds to residue Thr³⁹⁰ in a full-length, wild-type,human GSK3β amino add sequence, or corresponds so residue Ser³⁸⁹ in afull-length, wild-type, mouse GSK3β amino acid sequence. In someembodiments, the compound or procedure modulates expression or activityof a component of the p38 MAPK signaling pathway in the cell. In certainembodiments, the compound or procedure increases expression or activityof p38 MAPK. In some embodiments, the compound includes p38 MAPK. Insome embodiments, the disease or condition is a neurological disease orcondition. In some embodiment, the neurological disease or condition isAlzheimer's disease. In certain embodiments, the neurological disease orcondition is bipolar disorder. In some embodiments the neurologicaldisease or condition is stroke, cerebral ischemia, spinal cord trauma,head trauma, perinatal hypoxia, hypoglycemic neuronal damage, dementia(including AIDS-induced dementia), Alzheimer's disease, Huntington'sChorea, amyotrophic laterel sclerosis, multiple sclerosis, oculardamage, cognitive disorders, idiopathic and drug-induced Parkinson'sdisease, amyotrophic lateral sclerosis, tremors, epilepsy, convulsions,migraine (including migraine headache), psychosis, schizophrenia, mooddisorders (including depression, mania, bipolar disorders), trigeminalneuralgia, brain edema, pain (including acute and chronic pain states,severe pain, intractable pain, neuropathic pain, and post-traumaticpain), tardive dyskinesia, motor neuron disease, spinal muscularatrophy, progressive supranuclear palsy, or multiple sclerosis.

According to yet another aspect of the invention, methods for treating adisease or condition associated with GSK3β activity are provided. Themethods include administering to a subject having a disease or conditionassociated with GSK3β activity a therapeutically effective amount of acompound and/or application of a procedure that results in thephosphorylation of a GSK3β residue that corresponds to residue Thr³⁹⁰ ina full-length, wild-type, human GSK3β amino acid sequence, orcorresponds to residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3βamino acid sequence. In some embodiments, the compound or proceduremodulates expression or activity of a component of the p38 MAPKsignaling pathway in the cell. In certain embodiments, the compound orprocedure increases expression or activity of p38 MAPK. In someembodiments, the compound includes p38 MAPK. In some embodiments, thedisease or condition is a neurological disease or condition. In certainembodiments, the neurological disease or condition is Alzheimer'sdisease. In some embodiments, the neurological disease or condition isbipolar disorder. In some embodiments, the neurological disease orcondition is Alzheimer's disease. In some embodiments, the neurologicaldisease or condition is bipolar disorder. In certain embodiments theneurological disease or condition is stroke, cerebral ischemia, spinalcord trauma, head trauma, perinatal hypoxia, hypoglycemic neuronaldamage, dementia (including AIDS-induced dementia), Alzheimer's disease,Huntington's Chorea, amyotrophic lateral sclerosis, multiple sclerosis,ocular damage, cognitive disorders, idiopathic and drug-inducedParkinson's disease, amyotrophic lateral sclerosis, tremors, epilepsy,convulsions, migraine (including migraine headache), psychosis,schizophrenia, mood disorders (including depression, mania, bipolardisorders), trigeminal neuralgia, brain edema, pain (including acute andchronic pain states, severe pain, intractable pain, neuropathic pain,and post-traumatic pain), tardive dyskinesia, motor neuron disease,spinal muscular atrophy, progressive supranuclear palsy, or multiplesclerosis.

According to an aspect of the invention, methods of increasing activityof GSK3β in a cell are provided. The methods include contacting the cellwith a compound and/or applying a procedure to the cells that reducesphosphorylation of a residue that corresponds to residue Thr³⁹⁰ in afull-length, wild-type, human GSK3β amino acid sequence, or correspondsto residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3β amino acidsequence. In some embodiments, the compound or procedure modulatesexpression or activity of a component of the p38 MAPK signaling pathwayin the cell. In some embodiments, the compound or procedure reducesexpression or activity of p38 MAPK in the cell. In some embodiments, themethod is a method tor treating cancer.

According to yet another aspect of the invention, methods of treating adisease or condition associated with reduced GSK3β activity areprovided. The methods include administering to a subject having adisease or condition associated with reduced GSK3β activity atherapeutically effective amount of a compound and/or application of aprocedure of the cells that reduces phosphorylation of a residue thatcorresponds to residue Thr³⁹⁰ in a full-length, wild-type, human GSK3βamino acid sequence, or corresponds to residue Ser³⁸⁹ in a full-length,wild-type mouse GSK3β amino acid sequence. In certain embodiments, thecompound or procedure modulates expression or activity of a component ofthe p38 MAPK signaling pathway in the cell. In some embodiments, thecompound or procedure reduces expression or activity of p38 MAPK in thecell. In some embodiments, the disease or condition is cancer.

According to yet anther aspect of the invention, methods of identifyinga compound that modulates GSK3β activity are provided. The methodsinclude (a) contacting a GSK3β polypeptide of any of the aforementionedaspects of the invention that includes a phosphorylated residue thatcorresponds to residue Thr³⁹⁰ in a full-length, wild-type, human GSK3βamino acid sequence, with p38 MAPK and a putative modulating compoundunder suitable conditions for phosphorylation of the residue thatcorresponds to residue Thr³⁹⁰ in a lull-length, wild-type, human GSK3βamino acid sequence; (b) detecting the level of phosphorylation of theGSK3β residue that corresponds to residue Thr³⁹⁰ in a full-length,wild-type, human GSK3β amino acid sequence. In the contactedpolypeptide; (c) comparing the level of phosphorylation of the GSK3βresidue that corresponds to residue Thr³⁹⁰ in a full-length, wild-type,human GSK3β amino acid sequence. In the contacted polypeptide to a levelof phosphorylation of the corresponding residue in a control GSK3βpolypeptide not contacted with the compound, wherein if the level ofphosphorylation is higher in the contacted polypeptide than in thecontrol polypeptide the compound is identified as an Inhibitor of GSK3βactivity and if the level of phosphorylation is lower in the contactedpolypeptide than in the control polypeptide the compound is identifiedas an enhancer of GSK3β activity.

According to an aspect of the invention, methods of identifying acompound that modulates GSK3β activity are provided. The methods include(a) contacting a GSK3β polypeptide of any of the aforementioned aspectsof the invention that includes a phosphorylated residue that correspondsto residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3β amino acidsequence, with p38 MAPK and a putative modulating compound undersuitable conditions for phosphorylation of the residue that correspondsto residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3β amino acidsequence; (b) detecting the level of phosphorylation of the GSK3βresidue that corresponds to residue Ser³⁸⁹ in a full-length, wild-type,mouse GSK3β amino acid sequence. In the contacted polypeptide; and (c)comparing the level of phosphorylation of the GSK3β residue thatcorresponds to residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3βamino acid sequence, in the contacted polypeptide to a level ofphosphorylation of the corresponding residue in a control GSK3βpolypeptide not contacted with the compounds wherein if the level ofphosphorylation is higher in the contacted polypeptide than its thecontrol polypeptide the compound is identified as an inhibitor of GSK3βactivity and if the level of phosphorylation is lower in the contactedpolypeptide than in the control polypeptide the compound is identifiedas an enhancer of GSK3β activity.

According to one aspect of the invention, kits are provided. A kit ofthe invention may include any of the polypeptides of the aforementionedaspects of the invention.

BRIEF DESCRIPTION OF DRAWINGS

Fig. presents Western blots showing regulation of the β-catenin pathwayby p38 MAPK. FIG. 1A is a Western blot showing c-Myc and Lef in wholecell extracts from Rag^(−/−) thymocytes (Rag^(−/−)) and MKK6 thymocytes(MKK6). Actin was examined as a control. FIG. 1B is a Western blotshowing c-Myc and Lef in thymocytes from Rag^(−/−, MKK)6 andRag^(−/−)/MKK6 mice. FIG. 1C is a Western blot showing β-catenin innuclear extracts from Rag^(−/−) and MKK6 thymocytes. FIG. 1D is aWestern blot showing β-catenin and p38 MAPK in whole cell extracts from293T cells transfected with GSK3β (−) or GSK3β with p38 MAPK and MKK6(p38/MKK6).

Fig. presents Western blots showing direct phosphorylation of GSK3β byp38 MAPK. FIG. 2A is a Western blot showing phospho-Ser⁹ GSK3β (P-Ser⁹)and total GSK3β to Rag^(−/−) and MKK6 thymocytes. FIG. 2B is a Westernblot showing P-Ser⁹ GSK3β, GSK3β, phospho-p38 MAPK (P-p38) and p38 MAPKin 293T cells transfected with an empty vector (Con), MKK6 alone or MKK6and p38 MAPK (MKK6/p38). FIG. 2C is a Western blot showing results of anin vitro p38 MAP kinase assay with inactive recombinant GSK3β as thesubstrate, and p38 MAPK immunoprecipitated from MKK6-thymocytes (MKK6Thy) or MKK6-transfected 293T cells (293T). In vitro reactions wereincubated in the presence (SB) or absence (−) of the specific p38 MAPKinhibitor SB203580. Total GSK3β was visualized by PonceauS staining andphosphorylated GSK3β detected by autoradiography. FIG. 2D is a Westernblot showing results of an in vitro p38 MAPK kinase assay as describedin FIG. 2C using total or Akt-depleted extracts (Akt-dep) from MKK6thymocytes (MKK6 Thy) or MKK6-transfected 293T cells (293T). FIG. 2E isa Western blot showing results of an in vitro kinase assay as describedin FIG. 2C with recombinant active p38 MAPK kinase. FIG. 2F is a Westernblot showing GSK3β and p38 MAPK in the GSK3β and p21 (Con)immunoprecipitates (IP) and whole cell extracts from MKK6 thymocytes(Input). FIG. 2G is a Western blot showing results of an in vitro kinaseassay for recombinant active p38 MAPK kinase usingcatalytically-inactive GSK3β as a substrate. Phosphorylation of ATF2 wasexamined as a positive control.

Fig. presents Western blots and graphs showing that inhibition of GSK3βby p38 MAPK is mediated by phosphorylation at Thr³⁹⁰. FIG. 3A is aWestern blot showing results of an in vitro kinase assays forrecombinant p38 MAPK using catalytically-inactive GSK3β and GSK3β-T¹³Amutant as substrates. FIG. 3B is a Western blot showing results of an invitro kinase assay for recombinant p38 MAPK using kinase-inactive GSK3β(WT) GSK3β-T¹³A, GSK3β-T³⁹⁰A and GSK3β-T¹³A/T³⁹⁰A mutants as substrates.FIG. 3C is a Western blot showing results of an in vitro kinase assayfor recombinant active ERK using catalytically-inactive GSK3β,GSK3β-T¹³A, GSK3β-T¹³A and GSK3β-T¹³A/T¹³A mutants as substrates. FIG.3D-F present graphs showing results of an in vitro kinase assay foractive GSK3β, GSK3β-T¹³A and GSK3β-T³⁹⁰A mutants before (Con) or afterincubation with activated Akt or activated p38 MAPK. GSK3β activityrelative to the activity without Akt or p38 MAPK (Con) is shown. Errorbars represent SD (n=3). FIG. 3G is a graph showing results of in vitroGSK3β kinase reactions alone (−) or in the presence ofunphosphorylated-Thr³⁹⁰ (Thr³⁹⁰), phospho-Ser⁹ (P-Ser⁹), orphospho-Thr³⁹⁰ (P-Thr³⁹⁰) peptides as described in FIG. 3D-F. FIG. 3H-Jpresent graphs showing results of GSK3β in vitro kinase assays as inFIG. 3D-F using various concentrations of phospho-Thr³⁹⁰, phospho-Ser⁹and unphosphorylated-Thr³⁹⁰ peptides. Each point is the average of twomeasurements.

FIG. presents Western blots and graphs showing phosphorylation ofendogenous GSK3β by p38 MAPK. FIG. 4A is a Western blot showing thepresence of endogenous phospho-Ser³⁸⁹ GSK3β (P-Ser³⁸⁹) in wild-type,GSK3α^(−/−) and GSK3β^(−/−) ES cells. Total GSK3α, GSK3β and GAPDH wereexamined as controls. FIG. 4B is a Western blot showing P-Ser³⁸⁹ inGSK3β^(−/−)ES cells transfected with wild-type GSK3β or GSK3β-S³⁸⁹Amutant alone or with p38 MAPK and MKK6 (p38/MKK6). FIG. 4C is a Westernblot showing P-Ser³⁸⁹, Flag-tagged mouse GSK3β and β-catenin innon-transfected 293T cells (−) or cells transfected with mouse GSK3βalone or in combination with p38 MAPK and MKK6 (p38/MKK6). FIG. 4D is aWestern bios showing P-Ser³⁸⁹, total GSK3β and β-catenin in 293T cellstransfected with GSK3β, p38 and MKK6 in the absence (−) or presence ofSB203580. FIG. 4E-F present Western blots showing P-Ser³⁸⁹ and totalGSK3β in MEF or total GSK3α and GSK3β in ES cells non-treated (−) ortreated with SB203580. FIG. 4G-I present Western blots showing P-Ser³⁸⁹,P-Ser⁹, total GSK3β, P-p38, total p38 and β-catenin in WT andMKK3^(−/−)MKK6^(−/−) (3/6^(−/−)) MEF. FIG. 4J presents a Western blotshowing the tissue distribution of P-Ser³⁸⁹, P-Ser⁹ and total GSK3β.FIG. 4K presents a graph showing quantification of the levels ofP-Ser³⁸⁹ relative to P-Ser⁹ in each tissue. FIG. 4L presents Westernblots showing P-Ser³⁸⁹ and total GSK3β in thymocytes and brain from WTmice treated in vivo with vehicle (−) or SB203580 (SB). FIG. 4M is aWestern blot showing P-S³⁸⁹ and total GSK3β in thymocytes from Rag^(−/−)and MKK6 transgenic mice.

FIG. 5A-B present graphs showing that MKK6 transgenic thymocytes haveincreased expression of c-myc and lef mRNA. Gene expression was examinedby Affymetrix gene chip analysis using mRNA isolated from MKK6 andRag^(−/−) thymocytes. Fold increase of MKK6 c-myc and lef mRNA relativeto Rag^(−/−) mRNA are shown.

FIG. 6 presents a gel demonstrating through RT-PCR experiments that p38MAPK does not affect β-catenin mRNA levels. The mRNA levels of β-cateninand actin in non-transfected control (Con) 293T cells and cellstransiently transfected with expression constructs for GSK3β alone or incombination with p38 MAPK and MKK6 were examined by semi-quantitativeRT-PCR. Three serial ½ dilutions of cDNA were used to examine β-cateninand actin expression.

FIG. 7 presents a Western blot showing that p38 MAPK does not regulateAKT-mediated Ser⁹ phosphorylation of GSK3β. 293T cells were treated withvehicle control (Con), SB203580 (SB) or Wortmanin (Wort) for 40 minutes.The levels of phosphoro-Ser⁹ and GSK3β were determined by Western Blotanalysis.

FIG. 8 presents a Western blot showing Akt and p38 levels. Akt and p38MAPK were immunoprecipitated (IP) from whole cell extracts from MKK6thymocytes (Thy) or MKK6-transfected 293T cells (293).Immunoprecipitation of p21 was included as a negative control (Con).Total cell lysate (Input) from MKK6 thymocytes and theimmunoprecipitates were examined for Akt and p38 MAPK by Western blotanalysis.

FIG. 9 presents a Western blot showing that p38 MAPK associates withGSK3β. GSK3β was immunoprecipated from whole cell lysates from 293Tcells transiently transfected with expression constructs for GSK3β along(−),GSK3β and p38 MAPK (p38), or GSK3β, p38 MAPK and MKK6 (p38/MKK6).The levels of p38 and GSK3β in the immunoprecipitates were determined byWestern blot analysis.

FIG. presents MS/MS spectra identifying a TP motif-containingphosphopeptide IQAAASTPTNATAASDANTGDR (SEQ ID NO:97) at the C-terminusof GSK3β as a target for p38 MAPK. FIG. 10A-B show the b and y ionlayout for the middle of the peptide. Phosphorylation of serine-389(S³⁸⁹) will produce a h6 ion that is 80 Da heavier, whilephosphorylation of threonine-390 (T³⁹⁰) will produce a y16 ion that is80 Da heavier. These are the only ions that will differentiate thedifference in phosphorylation of S³⁸⁹ and T³⁹⁰. All other b and y ionswill be the same for both species. The b7, b8, y14, and y15 ions willdifferentiate between phosphorylation of T³⁹⁰ and T³⁹² (or higher).Similar b and y ions can be used to differentiate phosphorylations ofT³⁹⁵, etc. The tons actually identified as b and y ions are indicated inthe figure. There was no b6 ion present, but the y16 ion at m/z=1642.5and the presence of the y15 ion at m/z=1461.6 defines that thephosphorylation site is at T³⁹⁰ and not on any another serine orthreonine.

FIG. 11A-B present MS/MS spectra identifying a P-Thr⁴³Pro phosphopeptideVTTVVATPGQGPDRPQEVSTTDTK(SEQ ID NO:98) as a target for p38 MAPK asdescribed in FIG. 10.

FIG. 12 presents a graph showing that p38 MAPK does not inhibit GSK3αactivity in vitro. Recombinant kinase-active GSK3α was pre-incubated inthe reaction buffer alone (GSKα), with activated p38 MAPK or activatedAkt (15 min). In vitro GSK3α kinase assays were then performed using GSMas the substrate. Error bars represent SD (n=3).

FIG. 13 is a Western blot showing that the phospho-S³⁸⁹ Ab specificallyrecognized GSK3β phosphorylated at S³⁸⁹ by p38 MAPK. Recombinant mousewildtype GSK3β was incubated with or without recombinant active p38 MAPKfor 15 min, followed by an incubation with or without (−) CalfIntestinal Phosphatase (CIP). Recombinant GSK3β-S³⁸⁹A mutant was alsoincubated with active p38 MAPK as described for wildtype GSK3β, but nottreated with CIP. The presence of phospho-S³⁸⁹ was determined by Westernblot analysis using the anti-phosphro-S³⁸⁹ AB. Blots were re-probed fortotal GSK3β.

FIG. 14 is a Western blot showing phospho-Ser³⁸⁹ GSK3β and total GSK3βin whole-cell lysates from mouse heart and liver. GAPDH was examined asa loading control.

Fig. shows activated p38 MAPK in different tissues. FIG. 15A presents aWestern blot showing phospho-p38 (P-p38) and total p38 levels in wholecell extracts for thymocytes (Thy), splenocytes (Spl), kidney and brain.The level of GAPDH was examined as a loading control. FIG. 15B presentsa graph showing the ratio of phospho-p38 to total p38.

FIG. 16 represents a kit of the invention. The kit (10) shown in FIG. 16includes a set of containers for housing a compounds (12) or (14) suchas a phosphorylated GSK3β polypeptide or a composition that modulatesphosphorylation of GSK3β. As well as instructions (20). Additionalcomponents may also be included in the kit.

DETAILED DESCRIPTION

Aspects of the invention relate to compositions and methods forregulation of GSK3β activity. The invention is based at least in part onthe surprising discovery that p38 MAPK phosphorylates GSK3β at aC-terminal residue, distinct from the N-terminal GSK3β residuesphosphorylated by Akt. The C-terminal GSK3β residue phosphorylated byp38 corresponds to residue Thr³⁹⁰ in a full-length, wild-type, humanGSK3β amino acid sequence, or corresponds to residue Ser³⁸⁹ in afull-length, wild-type, mouse GSK3β amino acid sequence. Describedherein are GSK3β polypeptides containing phosphorylated residues andantibodies that recognize GSK3β polypeptides containing phosphorylatedresidues. Also described herein are methods for regulating GSK3βactivity through phosphorylation of the residue corresponding to residueThr³⁹⁰ in a full-length, wild-type, human GSK3β amino acid sequence, orcorresponding to residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3βamino acid sequence. Aspects of the invention further relate totreatment of disorders that are characterized by elevated or reducedGSK3β activity, through the use of phosphorylated GSK3β polypeptidesdescribed herein and through modulation of the p38 signaling pathway.Further described herein are screening methods to identify compoundsthat can modulate GSK3β activity.

Polypeptides

The present invention relates to phosphorylated GSK3β polypeptides. Awild-type, full-length human GSK3β polypeptide has the amino acidsequence set forth as Swissprot Accession No. P49841 (SEQ ID NO:1).According to aspects of the invention, a phosphorylated wild-type,full-length human GSK3β polypeptide also has the amino acid sequence setforth as Swissprot Accession No. P49841 but is phosphorylated at leaston a threonine residue corresponding to residue 390 of the full-lengthhuman GSK3β polypeptide, and designated herein as Thr³⁹⁰. A nucleic acidsequence encoding human wild-type, full-length GSK3β is set forth asGenbank Accession No. NM_002093 (SEQ ID NO:2). In some embodiments thephosphorylated GSK3β polypeptides are mouse polypeptides. A wild-type,full-length mouse GSK3β polypeptide has the amino acid sequence setforth as Genbank Accession No. NP_062801 (SEQ ID NO:3). According toaspects of the invention, a phosphorylated wild-type, full-length mouseGSK3β polypeptide also has the amino acid sequence set forth as Genbank,Accession No. NP_062801 but is phosphorylated at least on a serineresidue corresponding to residue 389 of the full-length mouse GSK3βpolypeptide, and designated herein as Ser³⁸⁹. The nucleic acid encodingmouse wild-type GSK3β polypeptide has GenBank Accession No. NM_019827and is set forth herein as SEQ ID NO:4. It will be understood that aGSK3β polypeptide of the invention may be phosphorylated at one or moreresidues in addition to being phosphorylated at the residuecorresponding to Thr³⁹⁰ or Ser³⁸⁹ of the human or mouse full-length,wild-type sequences, respectively.

The amino acid sequence of a non-phosphorylated, full-length, humanwild-type GSK3β polypeptide is set forth as SEQ ID NO: 1 and the aminoacid sequence of a Thr³⁹⁰-phosphorylated full-length, human wild-typeGSK3β polypeptide is provided as SEQ ID NO:5. The amino acid sequence ofa non-phosphorylated, full-length, mouse wild-type GSK3β polypeptide isset forth as SEQ ID NO:3 and the amino acid sequence of aSer³⁸⁹-phosphorylated full-length, mouse wild-type GSK3β polypeptide isprovided as SEQ ID NO:6.

The designation of a specific amino acid residue in a mutant or fragmentof GSK3β polypeptide is based on the corresponding residue identity in afull-length, wild-type GSK3β polypeptide. In some embodiments, more thanone residue in the GSK3β polypeptide is phosphorylated. In someembodiments, only one residue in the GSK3β polypeptide isphosphorylated. In certain embodiments, the GSK3β polypeptide is a humanpolypeptide and only a Thr³⁹⁰ residue is phosphorylated. In certainembodiments, the GSK3β polypeptide is a mouse polypeptide and only aSer³⁸⁹ residue is phosphorylated.

There may be allelic variation in GSK3β polypeptide sequences of theinvention including wild-type GSK3β polypeptide sequences and/or mutantGSK3β polypeptide sequences. As used herein, the term “allelic variant”means any of two or more alternative forms of a gene occupying the samechromosomal locus. Allelic variation arises naturally through mutation,and may result in polymorphism within populations. Gene mutations can besilent (no change in the encoded polypeptide) or may encode polypeptideswith altered amino acid sequences. An allelic variant of a polypeptideis a polypeptide encoded by an allelic variant of a gene. It will beunderstood by those of ordinary skill in the art that such allelicvariations may occur in full-length wild-type and mutant GSK3βpolypeptides and in fragments of wild-type and mutant polypeptides,GSK3β polypeptides of the invention may be allelic variants of wild-typeGSK3β or mutant GSK3β polypeptide sequences. One of ordinary skill inthe art will be able to identify how residues of variants of wild-typeand mutant GSK3β polypeptide correspond to residues of wild-type GSK3βpolypeptide using routine methods.

The invention, in some aspects, includes phosphorylated GSK3βpolypeptides. The term, “phosphorylated GSK3β polypeptide” means a GSK3βpolypeptide that has been phosphorylated at one or more residues. Insome embodiments of the invention, a GSK3β polypeptide may bephosphorylated at a threonine residue. In certain embodiments, a GSK3βpolypeptide may be phosphorylated only at the residue that correspondsto the Thr³⁹⁰ residue of wild-type, full-length human GSK3β polypeptide.In some embodiments, a phosphorylated GSK3β polypeptide is aphosphorylated GSK3β polypeptide that is phosphorylated at least at theamino acid residue that corresponds to the amino acid residue number 390of full-length wild-type human GSK3β polypeptide, which is set forthherein as SEQ ID NO:5. The residue in position 390 of wild-type,full-length GSK3β polypeptide is a threonine, and this threonine in thewild-type, full-length polypeptide and the residue that corresponds tothis position in fragments and in mutated forms of GSK3β may be referredto herein as “Thr³⁹⁰”. GSK3β in which at least the Thr³⁹⁰ residue isphosphorylated may be referred to herein as Thr³⁹⁰-phosphorylated GSK3β.As used herein the term “Thr³⁹⁰-phosphorylated GSK3β polypeptide” is aGSK3β polypeptide that is phosphorylated at least at the threonine thatcorresponds to the Thr³⁹⁰ residue of full-length, wild-type GSK3βpolypeptide.

In some embodiments of the invention, a GSK3β polypeptide may bephosphorylated at a serine residue. In certain embodiments, a GSK3βpolypeptide may be phosphorylated only at the residue that correspondsto the Ser³⁸⁹ residue of wild-type, full-length mouse GSK3β polypeptide.In some embodiments, a phosphorylated GSK3β polypeptide is aphosphorylated GSK3β polypeptide that is phosphorylated at least at theamino acid residue that corresponds to the amino acid residue number 389of full-length wild-type mouse GSK3β polypeptide, which is set forthherein as SEQ ID NO:6. The residue in position 389 of wild-type,full-length mouse GSK3β polypeptide is a serine, and this serine in thewild-type, full-length polypeptide and the residue that corresponds tothis position in fragments and in mutated forms of GSK3β may be referredto herein as “Ser³⁸⁹”. GSK3β in which the Ser³⁸⁹ residue isphosphorylated may be referred to herein as Ser³⁸⁹-phosphorylated GSK3β.As used herein the term “Ser³⁸⁹-phosphorylated GSK3β polypeptide” is aGSK3β polypeptide that is phosphorylated at least at the serine thatcorresponds to the Ser³⁸⁹ residue of full-length, wild-type GSK3βpolypeptide.

The use of nomenclature to describe the position of phosphorylatedresidues herein can be further exemplified with a fragment of afull-length human GSK3β polypeptide that includes a phosphorylatedthreonine residue. One such phosphorylated GSK3β polypeptide is setforth as RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7). A non-phosphorylated GSK3βpolypeptide having the same amino acid sequence as SEQ ID NO:7 is setforth as RIQAAASTPTN (SEQ ID NO:8). The threonine that is residue 8(Thr⁸) of SEQ ID NO:8 corresponds to the threonine that is residue 390(Thr³⁹⁰) of the wild-type, full-length human GSK3β polypeptide aminoacid sequence, thus the phosphorylated amino acid residue in SEQ ID NO:8may be referred to as the Thr8 residue of SEQ ID NO: 8, or as theresidue that corresponds to the Thr³⁹⁰ residue of full-length wild-typehuman GSK3β polypeptide. Those of ordinary skill in the art can readilydetermine the correspondence of a phosphorylated residue in a GSK3βpolypeptide sequence (wild-type or mutant) with a residue in afull-length, wild-type GSK3β polypeptide using routine sequencecomparison methods. One of ordinary skill in the art would be familiarwith the existence of different isoforms of proteins and how todetermine which amino acid residues are corresponding residues betweendifferent isoforms of the same protein, by sequence alignment.

As used herein with respect to polypeptides, proteins, or fragmentsthereof; “isolated” means separated from its native environment sodpresent in sufficient quantity to permit its identification or use.Isolated, when referring to a protein or polypeptide, means, forexample: (i) selectively produced by expression cloning, (ii) purifiedas by chromatography or electrophoresis, (iii) synthesized using one ormore synthetic methods, etc. isolated proteins or polypeptides may be,but need not be, substantially pure. The term “substantially pure” meansthat the proteins or polypeptides are essentially free of othersubstances with which they may be found in production, nature, or invivo systems to an extent practical and appropriate for their intendeduse. Substantially pure polypeptides may be obtained naturally orproduced using methods described herein and may be purified withtechniques well known in the art. Because an isolated protein may beadmixed with a pharmaceutically acceptable carrier in a pharmaceuticalpreparation, the protein may comprise only a small percentage by weighsof the preparation. The protein is nonetheless isolated in that it hasbeen separated from the substances with which it may be associated inliving systems, i.e. isolated from other proteins.

According to some aspects of the invention, fragments of full-length,wild-type or mutant GSK3β polypeptides are provided, fragments of theinvention are preferably fragments that retain a distinct functionalcapability of the polypeptide. Functional capabilities which can beretained in a fragment include interaction with antibodies, interactionwith other polypeptides or fragments thereof, kinase activity, etc.Polypeptide fragments can be synthesized using art-known methods, andtested for function using methods exemplified herein.

A fragment of a phosphorylated GSK3β polypeptide may comprise at least5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400 or419 (including each integer in between) contiguous amino acids of GSK3βpolypeptide having a consecutive sequence found in wild-type GSK3βpolypeptide or a modified GSK3β polypeptide sequence as describedherein. In some embodiments, a fragment includes a threonine residuethat corresponds to Thr³⁹⁰ of full-length, wild-type human GSK3βpolypeptide. A residue that corresponds to Thr³⁹⁰ may or may not bephosphorylated. In some embodiments, a fragment includes a serineresidue that corresponds to Ser³⁸⁹ of full-length, wild-type mouse GSK3βpolypeptide. A residue that corresponds to Ser³⁸⁹ may or may not bephosphorylated. Fragments of phosphorylated GSK3β polypeptide can beprepared using synthetic methods known in the art or may be naturalfragments of phosphorylated GSK3β polypeptides. Such fragments areuseful for a variety of purposes, including in the preparation ofmolecules that bind specifically to synthetic and naturallyphosphorylated GSK3β polypeptides and in immunoassays well known tothose of ordinary skill in the art, including competitive bindingimmunoassays.

Non-limiting examples of fragments of a human GSK3β polypeptide thatinclude a threonine that corresponds to the Thr³⁹⁰ of full-length,wild-type GSK3β wherein the residue that corresponds to the Thr³⁹⁰ offull-length, wild-type GSK3β is phosphorylated and is indicated byunderlining, are ASTPT (SEQ ID NO:9), ASTPTN (SEQ ID NO:10), ASTPTNA(SEQ ID NO:11), ASTPTNAT (SEQ ID NO:12), ASTPTNATA (SEQ ID NO:13),ASTPTNATAA (SEQ ID NO:14), ASTPTNATAAS (SEQ ID NO:15), AASTP (SEQ IDNO:16), AAASTP (SEQ ID NO:17) QAAASTP (SEQ ID NO:18), IQAAASTP (SEQ IDNO:19), RIQAAASTP (SEQ ID NO:20), ARIQAAASTP (SEQ ID NO:21), HARIQAAASTP(SEQ ID NO:22), AASTPTN (SEQ ID NO:23), AAASTPTN (SEQ ID NO:24),AASTPTNA (SEQ ID NO:25), QAAASTPTN (SEQ ID NO.26), AASTPTNAT (SEQ IDNO:27), IQAAASTPTN (SEQ ID NO:28), AASTPTNATA (SEQ ID NO:29),RISQAAASTPTN (SEQ ID NO:8), AASTPTNATAA (SEQ ID NO:30), ARIQAAASTPTN(SEQ ID NO:31), AASTPTNATAAS (SEQ ID NO:32), HARIQAAASTPTO (SEQ IDNO:33), AASTPTNATAASD (SEQ ID NO:34), QAAASTPTNA (SEQ ID NO:35),AAASTPTNAT (SEQ ID NO:36), IQAAASTPTNA (SEQ ID NO:37), AAASTPTNATA (SEQID NO:38), RIQAAASTPTNA (SEQ ID NO:39), and AAASTPTNATAA (SEQ ID NO:40).

Non-limiting examples of fragments of a mouse GSK3β polypeptide thatinclude a serine that corresponds so the Ser³⁸⁹ of full-length,wild-type GSK3β, wherein the residue that corresponds to the Ser³⁸⁹ offull-length, wild-type GSK3β is phosphorylated and is indicated byunderlining, are ASPPA (SEQ ID NO:41), ASPPAN (SEQ ID NO:42), ASPPANA(SEQ ID NO:43), ASPPANAT (SEQ ID NO:44), ASPPANATA (SEQ ID NO:45),ASPPANATAA (SEQ ID NO:46), ASPPANATAAS (SEQ ID NO:47), AAASP (SEQ IDNO:48), QAAASP (SEQ ID NO:49), IQAAASP (SEQ ID NO:50), RIQAAASP (SEQ IDNO:51), ARIQAAASP (SEQ ID NO:52), HARIQAAASP (SEQ ID NO:53), PHARIQAAASP(SEQ ID NO:54), AASPP (SEQ ID NO:55), AAASPP (SEQ ID NO:56), AASPPA (SEQID NO:57), QAAASPP (SEQ ID NO:58), AASPPAN (SEQ ID NO:59), IQAAASPP (SEQID NO:60), AASPPANA (SEQ ID NO:61), RIQAAASPP (SEQ ID NO:62), AASPPANAT(SEQ ID NO:63), ARIQAAASTP (SEQ ID NO:64), AASPPANATA (SEQ ID NO:65),HARIQAAASPP (SEQ ID NO:66), AASPPANATAA (SEQ ID NO:67), PHARIQAAASPP(SEQ ID NO:68), AASPPANATAAS (SEQ ID NO:69), AAASPPA (SEQ ID NO:70),QAAASPPA (SEQ ID NO:71), AAASPPAN (SEQ ID NO:72), IQAAASPPA (SEQ IDNO:73), AAASPPANA (SEQ ID NO:74), RIQAAASPPA (SEQ ID NO:75), AAASPPANAT(SEQ ID NO:76), ARIQAAASPPA (SEQ ID NO:77), AAASPPANATA (SEQ ID NO:78),HARIQAAASPPA (SEQ ID NO:79), AAASPPANATAA (SEQ ID NO:80), ARIQAAASPPAN(SEQ ID NO: 81), QAAASPPANATA (SEQ ID NO:82), ARIQAAASPPANA (SEQ IDNO:83), IQAAASPPANATA (SEQ ID NO:84), ARIQAAASPPANAT (SEQ ID NO:85),RIQAAASPPANATA (SEQ ID NO:86), ARIQAAASPPANATA (SEQ ID NO:87),ARIQAAASPPANATA (SEQ ID NO:88), ARIQAAASPPANATAA (SEQ ID NO:89), andHARIQAAASPPANATA (SEQ ID NO:90).

One of ordinary skill in the art will understand how to prepareadditional fragments of full-length wild-type or mutant GSK3βpolypeptide. A phosphorylated fragment of a full-length wild-type ormutant human GSK3β polypeptide may includes phosphorylated threoninethat corresponds to the Thr³⁹⁰ threonine of full-length wild-type humanGSK3β polypeptide. It should be appreciated that a GSK3β polypeptidethat contains a phosphorylated Thr³⁹⁰ residue may or may not containother residues that are also phosphorylated. A phosphorylated fragmentof a full-length wild-type or mutant mouse GSK3β polypeptide may includea phosphorylated serine that corresponds to the Ser³⁸⁹ serine offull-length wild-type mouse GSK3β polypeptide. It should be appreciatedthat a GSK3β polypeptide that contains a phosphorylated Ser³⁸⁹ residuemay or may not contain other residues that are also phosphorylated.

One of ordinary skill in the art will recognize that a GSK3β polypeptidefragment that includes a threonine residue that corresponds to Thr³⁸⁹ offull-length, wild-type human GSK3β polypeptide may be a polypeptide thatincludes a threonine residue that corresponds to the Thr³⁹⁰ residue offull-length, wild-type GSK3β polypeptide with an additional 2, 3, 4, 5,6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209,210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223,224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307,308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321,322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349,350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363,364, 365, 366, 367, 360, 369, 370, 371, 372, 373, 374, 375, 376, 377378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,392, 392, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417 or 418 aminoacids, including all integers up to the sequence of a full-lengthwild-type or mutant, human GSK3β polypeptide minus one amino acid. Insome embodiments the GSK3β polypeptide is a full-length polypeptide. Theadditional amino acids may be added to either and/or both the N-terminusor the C-terminus of the threonine that corresponds to a Thr³⁹⁰ aminoacid, such that the amino acid sequence corresponds to an amino acidsequence of a wild-type or mutant human GSK3β polypeptide, or a modifiedwild-type or mutant human GSK3β polypeptide.

In some embodiments a GSK3β polypeptide fragment includes a serineresidue that corresponds to Ser³⁹⁰ of full-length, wild-type mouse GSK3βpolypeptide may be a polypeptide that includes a serine residue thatcorresponds to the Ser³⁸⁹ residue of full-length, wild-type GSK3βpolypeptide with an additional 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 90, 100, 101, 102,103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116,117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172,173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200,201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270,271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284,285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298,299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354,355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368,369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382,383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 392, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,411, 412, 413, 414, 415, 416, 417 or 418 amino acids, including allintegers up to the sequence of a full-length wild-type or mutant mouseGSK3β polypeptide minus one amino acid. In some embodiments the GSK3βpolypeptide is a full-length polypeptide. The additional amino acids maybe added to either and/or both the N-terminus or the C-terminus of theserine that corresponds to a Ser³⁸⁹ amino acid, such that the amino acidsequence corresponds to an amino acid sequence of a wild-type or mutantmouse GSK3β polypeptide, or a modified wild-type or mutant mouse GSK3βpolypeptide. One of ordinary skill in the art would be aware thatfunctional homologs of human and/or mouse GSK3β exist in multiplespecies. Polypeptides including full-length proteins and fragments offull-length proteins from other species, that are functionallyhomologous to human and/or mouse GSK3β are compatible with the instantinvention. One of ordinary skill in the art would further be aware oftechniques to identify a residue in a homologous protein that isfunctionally homologous to residue Thr³⁹⁸ in human GSK3β and/or residueSer³⁸⁹ in mouse GSK3β.

A “modified” wild-type or mutant GSK3β polypeptide or fragment thereofmay include deletions, point mutations, truncations, amino acidsubstitutions and/or additions of amino acids or non-amino acidmoieties. Modifications of a polypeptide of the invention may be made bymodification of the nucleic acid that encodes the polypeptide oralternatively, modifications may be made directly to the polypeptide,such as by cleavage, addition of a linker molecule, addition of adetectable moiety, such as biotin, addition of a carrier molecule, andthe like. Modifications also embrace fusion proteins comprising all orpart of the polypeptide's amino acid sequence.

In general, modified GSK3β polypeptides include polypeptides that aremodified specifically to alter a mature of the polypeptide unrelated toits physiological activity. For example, cysteine residues can besubstituted or deleted to prevent unwanted disulfide linkages.Polypeptide modifications can be made by selecting an amino acidsubstitution, deletion, and/or addition, and a modified polypeptide maybe synthesized using art-known methods/ Modified polypeptides then canbe tested for one or more activities (e.g., kinase activity, antibodybinding, antigenicity, ability to interact with a substrate, etc.) todetermine which modification provides a modified polypeptide with thedesired properties.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in a polypeptide to provide functionallyequivalent polypeptides, i.e., modified GSK3β polypeptides that retain afunctional capability of a wild-type or mutant GSK3β polypeptide. Asused herein, a “conservative amino acid substitution” refers to an aminoacid substitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Modified GSK3β polypeptides can be prepared according to methodsfor altering polypeptide sequence and known to one of ordinary skill inthe art such. Exemplary functionally equivalent GSK3β polypeptidesinclude conservative amino acid substitutions of an GSK3β polypeptide,or fragments thereof, such as a modified GSK3β polypeptide. Conservativesubstitutions of amino acids include substitutions made amongst aminoacids within the following groups: (a) M, I L, V; (b) F, Y, W; (c) K, R,H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Without wishing to be bound by any theory or mechanism, in someembodiments, phosphorylated GSK3β polypeptides of the invention mayinhibit the activity of GSK3β by binding to the catalytic site on theenzyme and competing with internal substrates.

Conservative amino-acid substitutions in a GSK3β polypeptide typicallyare made by alteration of a nucleic acid encoding the polypeptide. Suchsubstitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis, or by chemicalsynthesis of a gene encoding the GSK3β polypeptide. Where amino acidsubstitutions are made to a small fragment of a polypeptide, thesubstitutions can be made by directly synthesizing the polypeptide. Theactivity of functionally equivalent fragments of GSK3β polypeptides canbe tested by cloning the gene encoding the altered polypeptide into abacterial or mammalian expression vector, introducing the vector into anappropriate host cell, expressing the altered polypeptide, and testingfor a functional capability of the polypeptide as disclosed herein.

As described above, a fragment of a full-length wild-type or mutantGSK3β polypeptide may be a synthetic polypeptide. As used herein, theterm “synthetic” means artificially prepared. A synthetic polypeptide isa polypeptide that is synthesized and is not a naturally producedpolypeptide molecule (e.g., not produced in an animal or organism), itwill be understood that the sequence of a natural polypeptide (e.g., anendogenous polypeptide) may be identical to the sequence of a syntheticpolypeptide, but the latter will have been prepared using at least onesynthetic step.

As used herein, a synthetic phosphorylated polypeptide is a polypeptidephosphorylated with a synthetic method, which may be, but is not limitedto a method of the invention. A phosphorylated polypeptide of theinvention may be a naturally phosphorylated polypeptide (e.g., anaturally phosphorylated polypeptide) or may be a syntheticphosphorylated polypeptide. Although a synthetic, phosphorylatedpolypeptide may differ from a natural phosphorylated polypeptide, anantibody raised against a synthetic polypeptide of the invention willspecifically bind the synthetic polypeptide epitope against which it wasraised, and will also specifically bind the natural epitope in apolypeptide. Thus, even though a phosphorylated epitope of a syntheticpolypeptide may differ slightly in amino acid sequence from the sameepitope in a natural phosphorylated polypeptide, an antibody raisedagainst a synthetic phosphorylated epitope of the invention specificallybinds, in some cases, with high affinity to the natural phosphorylatedepitope and to a synthetic phosphorylated epitope. Antibodies of theinvention generated using a synthetic phosphorylated polypeptidespecifically bind, in some cases, with high affinity so natural andsynthetic phosphorylated polypeptides and are able to distinguishbetween natural (heterogeneous) phosphorylated and naturalnon-phosphorylated polypeptides and also to distinguish betweensynthetic phosphorylated and synthetic non-phosphorylated polypeptides.

Antibodies

The invention includes in one aspect, methods and compositions forpreparing antibodies that specifically bind synthetic and naturalphosphorylated GSK3β. The invention includes, in part, methods forpreparing phosphorylated GSK3β polypeptides, including, but not limitedto Thr³⁹⁰-phosphorylared human GSK3β polypeptides orSer³⁸⁹-phosphorylated mouse GSK3β polypeptides. Phosphorylated GSK3βpolypeptides may be used as antigens to make antibodies thatspecifically bind phosphorylated GSK3β polypeptide. Compositions usefultor making an antibody of the invention may include a phosphorylatedGSK3β polypeptide. In embodiments of the invention, a phosphorylatedGSK3β polypeptide or fragment thereof may be a phosphorylatedfull-length, wild-type or mutant GSK3β polypeptides or a fragment of awild-type or mutant full-length GSK3β that is a phosphorylated fragment.

Methods of the invention may also include the use of fragments of GSK3βpolypeptides for the production of antibodies that specifically bindphosphorylated GSK3β polypeptides. In some embodiments, a phosphorylatedthreonine residue of a GSK3β polypeptide that is part of the epitopespecifically recognized by the antibody is a threonine residue thatcorresponds to a phosphorylated residue of wild-type, full-length humanGSK3β polypeptide. In certain embodiments, a phosphorylated residuecorresponds to residue Thr³⁹⁰ of wild-type, full-length human GSK3βpolypeptide. In some embodiments, a phosphorylated serine residue of aGSK3β polypeptide that is part of the epitope specifically recognized bythe antibody is a serine residue that corresponds to a phosphorylatedresidue of wild-type, full-length mouse GSK3β polypeptide. In someembodiments, a phosphorylated residue corresponds to residue Ser³⁸⁹ ofwild-type, full-length mouse GSK3β polypeptide. In some embodiments, anantigenic polypeptide can be as small as 5 amino acids in length. Forexample, ASTPT(SEQ ID NO:9), ASTPTN (SEQ ID NO: 10), ASTPTNA (SEQ ID NO:11), ASTPTNAT (SEQ ID NO:12), ASTPTNATA (SEQ ID NO:13), ASTPTNATAA (SEQID NO:14), ASTPTNATAAS (SEQ ID NO:15), AASTP (SEQ ID NO:16), AAASTP (SEQID NO:17), QAAASTP (SEQ ID NO:18), IQAAASTP (SEQ ID NO:19), RIQAAASTP(SEQ ID NO:20), ARIQAAASTP (SEQ ID NO:21), HARIQAAASTP (SEQ ID NO:22);AASTPTN (SEQ ID NO:23), AAASTPTN (SEQ ID NO:24), AASTPTNA (SEQ IDNO:25), QAAASTPTN (SEQ ID NO:26), AASTPTNAT (SEQ ID NO:27), IQAAASTPTN(SEQ ID NO:28), AASTPTNATA (SEQ ID NO:29), RIQAAASTPTN (SEQ ID NO:8),AASTPTNATAA (SEQ ID NO:30), ARIQAAASTPTN (SEQ ID NO:31), AASTPTNATAAS(SEQ ID NO:32), HARIQAAASTPTN (SEQ ID NO:33), AASTPTNATAASD (SEQ IDNO:34), QAAASTPTNA (SEQ ID NO:35), AAASTPTNAT (SEQ ID NO:36),IQAAASTPTNA (SEQ ID NO:37), AAASTPTNATA (SEQ ID NO:38), RIQAAASTPTNA(SEQ ID NO:39), and AAASTPTNATAA (SEQ ID NO:40) are non-limitingexamples of phosphorylated antigenic fragments that may be used togenerate antibodies that specifically recognize a Thr³⁹⁰-phosphorylatedhuman GSK3β polypeptide, wherein the underlined residue represents theresidue that corresponds to Thr³⁹⁰ and is phosphorylated.

ASPPA (SEQ ID NO:41), ASPPAN (SEQ ID NO:42), ASPPANA (SEQ ID NO:43),ASPPANAT (SEQ ID NO:44), ASPPANATA (SEQ ID NO:45), ASPPANATAA (SEQ IDNO:46), ASPPANATAAS (SEQ ID NO:47), AAAST (SEQ ID NO:48), QAAASP (SEQ IDNO:49), IQAAASP (SEQ ID NO:50), RIQAAASP (SEQ ID NO:51), ARIQAAASP (SEQID NO:52), HARIQAAASP (SEQ ID NO:53), PHARIQAAASP (SEQ ID NO:54), AASPP(SEQ ID NO:55), AAASPP (SEQ ID NO:56), AASPPA (SEQ ID NQ:57), QAAASPP(SEQ ID NO:58), AASPPAN (SEQ ID NO:59), IQAAASPP (SEQ ID NO:60),AASPPANA (SEQ ID NO:61), RIQAAASPP (SEQ ID NO:62), AASPPANAT (SEQ IDNO:63), ARIQAAASPP (SEQ ID NO:64), AASPPANATA (SEQ ID NO:65),HARIQAAASPP (SEQ ID NO:66), AASPPANATAA (SEQ ID NO:67), PHARIQAAASPP(SEQ ID NO:68), AASPANATAAS (SEQ ID NO:69), AAASPPA (SEQ ID NO:70),QAAASPPA (SEQ ID NO:71), AAASPPAN (SEQ ID NO:72), IQAAASPPA (SEQ IDNO:73), AAASPPANA (SEQ ID NO:74), RIQAAASPPA (SEQ ID NO:75), AAASPPANAT(SEQ ID NO:76), ARIQAAASPPA (SEQ ID NO:77), AAASPPANATA (SEQ ID NO:78),HARIQAAASPPA (SEQ ID NO:79), AAASPPANATAA (SEQ ID NO:80), ARIQAAASPPAN(SEQ ID NO:81), QAAASPPANATA (SEQ ID NO:82), ARIQAAASPPANA (SEQ IDNO:83), IQAAASPPANATA (SEQ ID NO:84), ARIQAAASPPANAT (SEQ ID NO:85),RIQAAASPPANATA (SEQ ID NO:86), ARIQAAASPPANATA (SEQ ID NO:87),ARIQAAASPPANATA (SEQ ID NO:88), ARIQAAASPPANATAA (SEQ ID NO.89), andHARIQAAASPPANATA (SEQ ID NO:90), are non-limiting examples ofphosphorylated antigenic fragments that may be used to generateantibodies that specifically recognize a Ser³⁸⁹-phosphoryated mouseGSK3β polypeptide, wherein the underlined residue represents the residuethat corresponds to Ser³⁸⁹ and is phosphorylated. In some embodiments,when the size of the polypeptide antigen is less than about 8 aminoacids in length, a second carrier molecule, e.g., bovine serum albumin(BSA), may be attached to the polypeptide to increase antigenicity ofthe polypeptide. Thus, small fragments of GSK3β that include the desiredepitope for antibody production can be used in the production of anantibody that specifically binds to the epitope, which includes aphosphorylated threonine residue (e.g., a Thr³⁹⁰-phosphorylated residueor a Ser³⁸⁹-phosphorylated residue).

In some embodiments, antibodies that specifically bindARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87), are provided. In certainembodiments the antibody that specifically binds toARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87) is phospho-S³⁸⁹ GSK3β. In thepreparation of antibodies that specifically bind toSer³⁸⁹-phosphorylated GSK3β, ARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87) orother GSK3β polypeptide fragments that include a phosphorylated Ser³⁸⁹residue may be used. In some embodiments, antibodies that specificallybind RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7) are provided. In the preparationof antibodies that specifically bind to Thr³⁹⁰-phosphorylated GSK3β,RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7) or other GSK3β polypeptide fragmentsthat include a phosphorylated Thr³⁹⁰ residue may be used. Any GSK3βpolypeptide fragment that includes a phosphorylated serine or threonineresidue may be used in conjunction with a second molecule, e.g., keyholelimpet hematocyanin (KLH) or bovine serum albumin (BSA) as describedabove, as an antigenic polypeptide with which to prepare antibodies thatspecifically bind to a phosphorylated GSK3β polypeptide. In someembodiments, an antigenic polypeptide may be a polypeptide fragment thatincludes phosphorylated Thr³⁹⁰, and an antibody generated from such mantigen will specifically bind to a Thr³⁹⁰-phosphorylated epitope ofGSK3β polypeptide. In some embodiments, an antigenic polypeptide may bea GSK3β polypeptide fragment that includes phosphorylated Ser³⁸⁹, and anantibody generated from such an antigen will specifically bind to aSer³⁸⁹-phosphorylated epitope of GSK3β polypeptide. Anti-GSK3βpolypeptide antibodies or antigen-binding fragments thereof may bepurified using art-known affinity purification and/or affinity selectionmethods. Affinity selection is selection of antibodies orantigen-binding fragments thereof for binding to the target material(e.g., a phosphorylated GSK3β polypeptide).

It will be understood by those of ordinary skill in the art that it ispreferable that a fragment of GSK3β polypeptide for use as animmunogenic fragment in the methods of the invention be at least 5, 6, 78, 9, 10, 11, 12, 13, 14, 15, 16, 18, 18, 19, 20 or more amino acids inlength. In some embodiments if a fragment of GSK3β polypeptide includesmore than one threonine residue, it is desirable that only one of thethreonine residues is a phosphorylated threonine residue. In someembodiments if a fragment of GSK3β polypeptide includes more than oneserine residue. It is desirable that only one of the serine residues isa phosphorylated serine residue. One of ordinary skill in the art willbe able to use the guidance provided herein to make additional fragmentsof GSK3β polypeptide that can be used in methods of the invention.

As used herein, the term “antibody” refers to a glycoprotein that mayinclude at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or V_(H)) and aheavy chain constant region. The heavy chain constant region iscomprised of three domains, C_(H)1, C_(H)2 and C_(H)3. Each light chainis comprised of a light chain variable region (abbreviated herein asLCVR or V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, CL. The V_(H) and V_(L)regions can be farther subdivided into regions of hyper variability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). EachV_(H) and V_(L) is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system.

The term “antigen-binding fragment” of an antibody as used herein,refers to one or more portions of an antibody that retain the ability tospecifically bind to an antigen (e.g., phosphorylated GSK3βpolypeptide). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding fragment” of an antibody include (i) a fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H)Idomains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and CH1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546)which consists of a V_(H) domain: and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded tor by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv); see e.g., Bird et al (1988) Science 242:423-426; and Huston etal. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional procedures, such as proteolyticfragmentation procedures, as described in J. Goding, MonoclonalAntibodies: Principles and Practice, pp 98-118 (N.Y. Academic Press1983), which is hereby incorporated by reference as well as by othertechniques known to those with skill in the art. The fragments arescreened for utility in the same manner as are intact antibodies.

Isolated antibodies of the invention encompass various antibodyisotypes, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA sec, IgD,IgE. As used herein, “isotype” refers to the antibody class (e.g., IgMor IgG1) that is encoded by heavy chain constant region genes.Antibodies of the invention can be full length or can include only anantigen-binding fragment such as the antibody constant and/or variabledomain of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgA sec, IgD or IgEor could consist of a Fab fragment, a F(ab′)₂ fragment, and a Fvfragment.

Antibodies of the present invention can be polyclonal, monoclonal, or amixture of polyclonal and monoclonal antibodies. Antibodies of theinvention can be produced by-methods disclosed herein or by a variety oftechniques known in the art. In some embodiments, the epitope recognizedby an antibody of the invention comprises a phosphorylated threoninethat corresponds to the Thr³⁹⁰ in full-length, wild-type human GSK3βpolypeptide. In some embodiments, the epitope recognized by an antibodyof the invention comprises a phosphorylated residue that corresponds toSer³⁸⁹ of wild-type, full-length mouse GSK3β polypeptide.

Polyclonal and monoclonal antibodies may be prepared using techniquesthat are known in the art. The term “monoclonal antibody,” as usedherein, refers to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody displays a single bindingspecificity and affinity for a particular epitope. The term “polyclonalantibody” refers to a preparation of antibody molecules that comprises amixture of antibodies active that specifically bind a specific antigen.

A process of monoclonal antibody production may include obtaining immunesomatic sells with the potential for producing antibody, in particular Blymphocytes, which have been previously immunized with the antigen ofinterest either in vivo or in vitro and that are suitable for fusionwith a B-cell myeloma line. Mammalian lymphocytes typically areimmunized by in vivo immunization of the animal (e.g., a mouse or othermammal) with the desired protein or polypeptide, e.g., withphosphorylated GSK3β polypeptide or a fragment thereofThr³⁹⁰-phosphorylated GSK3β or a fragment thereof, orSer³⁸⁹-phosphorylated GSK3β or a fragment thereof, in the presentinvention. In some embodiments, the polypeptide is a modifiedpolypeptide as described herein. In some embodiments the polypeptidecomprises the sequence set forth as SEQ ID NO:7. Such immunizations arerepeated as necessary at intervals of up to several weeks to obtain asufficient titer of antibodies. Once immunized, animals can be used as asource of antibody-producing lymphocytes. Following the last antigenboost, the animals are sacrificed and spleen cells removed. Mouselymphocytes give a higher percentage of stable fissions with the mousemyeloma lines described herein. Of these, the SALB/c mouse is preferred.However, other mouse strains, rat, rabbit, hamster, sheep, goats,camels, llamas, frogs, etc. may also be used as hosts for preparingantibody-producing cells. (See; Goding in Monoclonal Antibodies:Principles and Practice, 2d ed., pp. 60-61, Orlando, Fla., AcademicPress, 1986). Mouse strains that have human immunoglobulin genesinserted in the genome (and that cannot produce mouse immunoglobulins)can also be used. Examples include the HuMAb mouse strains produced byMedarex/GenPharm International, and the XenoMouse strains produced byAbgenix. Such mice produce fully human immunoglobulin molecules inresponse to immunization.

Those antibody-producing cells that are in the dividing plasmablaststage fuse preferentially. Somatic cells may be obtained from the lymphnodes, spleens and peripheral blood of antigen-primed animals, and thelymphatic cells of choice depend to a large extent on their empiricalusefulness in the particular fusion system. The antibody-secretinglymphocytes are then fused with B cell myeloma cells or transformedcells, which are capable of replicating indefinitely in cell culture,thereby producing an immortal, immunoglobulin-secreting cell line. The Bcell myeloma cells or transformed cells may be mouse or other suitablemammalian cells. The resulting fused cells, or hybridomas are cultured,and the resulting colonies screened for the production of the desiredmonoclonal antibodies. Colonies producing such antibodies are cloned,and grown either in vivo or in vitro to produce large quantities ofantibody. A description of the theoretical basis and practicalmethodology of fusing such cells is set forth in Kohler and Milstein,Nature 256:495 (1975), which is hereby incorporated by reference.

Myeloma cell lines suited for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzymes deficiencies that render them incapable ofgrowing in certain selective media which support the growth of thedesired hybridomas. Examples of such myeloma cell lines that may be usedfor the production of fused cell lines include, but are not limited toAg8, P3-X63/Ag8, X63-Ag8.653, NSI/1.Ag 4.1, Sp2/0-Ag14, FO, NSO/U,MPC-11, MPC11-X4S-GTG 1.7, S194/SXX0 Bul, all derived from mice;R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210 derived from rats and U-266,GMI500-GRG2, LICR-LON-HMy2, UC729-6, all derived from humans (Goding, inMonoclonal Antibodies: Principles and Practice, 2d ed, pp. 65-66,Orlando, Fla., Academic Press, 1986; Campbell, in Monoclonal AntibodyTechnology, Laboratory Techniques in Biochemistry and Molecular BiologyVol. 13, Burden and Von Knippenberg, eds. pp. 75-83, Amsterdam,Elsevier, 1984). Those of ordinary skill in the art will be aware ofnumerous routine methods to produce monoclonal antibodies.

Fusion with mammalian myeloma cells or other fission partners capable ofreplicating indefinitely in cell culture is effected by standard andwell-known techniques, for example, by using polyethylene glycol (“PEG”)or other (using agents (See Milstein and Kohler, Eur. J. Immunol. 6:511(1976), which is hereby incorporated by reference).

Methods for raising polyclonal antibodies are well known to those ofordinary skill in the art. As a non-limiting example,anti-phosphorylated GSK3β polyclonal antibodies may be raised byadministering a phosphorylated GSK3β polypeptide subcutaneously toNew-Zealand white rabbits which have first been bled to obtainpre-immune serum. The phosphorylated GSK3β can be inoculated with (e.g.,injected at) a total volume of 100 μl per site at six different sites,typically with one or more adjuvants. The rabbits are then bled twoweeks after the first injection and periodically boosted with the sameantigen three times every six weeks. A sample of serum is collected 10days after each boost. Polyclonal antibodies are recovered from theserum, preferably by affinity chromatography using phosphorylated GSK3βto capture the antibody. This and other procedures for raisingpolyclonal antibodies are disclosed in E. Harlow, et al., editors,Antibodies: A Laboratory Manual (1988), which is hereby incorporated byreference. Those of ordinary skill in the art will be aware of numerousroutine methods to produce polyclonal antibodies. In some embodiments,the epitope recognized by the polyclonal antibody of the inventioncomprises a phosphorylated residue that corresponds to Thr³⁹⁰ ofwild-type, full-length human GSK3β polypeptide. In some embodiments, theepitope recognized by the polyclonal antibody of the invention comprisesa phosphorylated residue that corresponds to Ser³⁸⁹ of wild-type,full-length mouse GSK3β polypeptide.

In other embodiments, antibodies may be recombinant antibodies. The term“recombinant antibody”, as used herein, is intended to includeantibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse, rat, rabbit, etc.) that is transgenic for another species'immunoglobulin genes, genetically engineered antibodies, antibodiesexpressed using a recombinant expression vector transfected into a hostcell, antibodies isolated from a recombinant, combinatorial antibodylibrary, or antibodies prepared, expressed, created or isolated by anyother means that involves splicing of immunoglobulin gene sequences toother DNA sequences.

The present invention further provides nucleic acid molecules encodinganti-phosphorylated GSK3β antibodies (e.g., anti-Thr³⁹⁰-phosphorylatedGSK3β antibodies or anti-Ser³⁸⁹-phosphorylated GSK3β antibodies) andvectors comprising the nucleic acid molecules as described herein. Thevectors provided can be used to transform or transfect host cells forproducing anti-phosphorylated GSK3β antibodies with the specificity ofantibodies described herein. In some embodiments the antibodies producedwill have the specificity of the phospho-S³⁸⁹ GSK3β antibody. In someembodiments, the vectors can include an isolated nucleic acid moleculeencoding a heavy chain and/or a light chain of an antibody of theinvention encoded by a nucleic acid molecule. In a further embodiment,plasmids are given which produce the antibodies or antigen-bindingfragments described herein.

Antibodies or antigen-binding fragments of the invention are,preferably, isolated. “Isolated”, as used herein with respect toantibodies and antigen-binding fragments thereof, is intended to referto an antibody (or antigen-binding fragment thereof) that issubstantially free of other antibodies (or antigen-binding fragments)having different antigenic specificities (e.g., an isolated antibodythat specifically binds to phosphorylated GSK3β polypeptide issubstantially free of antibodies that specifically bind antigens otherthan phosphorylated GSK3β polypeptide). An isolated antibody thatspecifically binds to an epitope, isoform or variant of a phosphorylatedpolypeptide (e.g., phosphorylated GSK3β polypeptide) may, however, havecross-reactivity to other related antigens, e.g., a mutant form ofGSK3β, or a polypeptide from other species (e.g., GSK3β specieshomologs). Moreover, an isolated antibody (or antigen-binding fragmentthereof) may be substantially free of other cellular material and/orchemicals.

Antibodies of the invention include, but are not limited to antibodiesthat specifically bind to a phosphorylated GSK3β polypeptide. In certainembodiments, an antibody of the invention specifically binds GSK3β thatis phosphorylated at a residue that corresponds to the Thr³⁹⁸ residue offull-length, wild-type human GSK3β polypeptide, in certain embodiments,an antibody of the invention specifically binds GSK3β that isphosphorylated at a residue that corresponds to the Ser³⁸⁹ residue offull-length, wild-type mouse GSK3β polypeptide. As used herein,“specific binding” refers to antibody binding to a predetermined antigenwith a preference that enables the antibody to be used to distinguishthe antigen from others to an extent that permits the diagnostic andother assays described herein. For example specific binding toThr³⁹⁰-phosphorylated GSK3β polypeptide means that the antibody not onlypreferentially binds GSK3β polypeptide versus other polypeptides, butalso that it preferentially binds a phosphorylated GSK3β polypeptideversus a GSK3β polypeptide that is not phosphorylated. The antibody maybind with an affinity that is at least two-fold greater than itsaffinity for binding to antigens other than the predetermined antigen.In some embodiments, an antibody or antigen-binding fragment thereof ofthe invention specifically binds to Thr³⁹⁰-phosphorylated GSK3βpolypeptide. In some embodiments, an antibody or antigen-bindingfragment thereof of the invention specifically binds toSer³⁸⁹-phosphorylated GSK3β polypeptide. It will be understood that theGSK3β polypeptide or fragment thereof that includes a phosphorylatedresidue that corresponds to phosphorylated Thr³⁹⁰ of full-length,wild-type human GSK3β polypeptide, or phosphorylated Ser³⁸⁹ offull-length, wild-type mouse GSK3β polypeptide may be a wild-type or amutant form of GSK3β polypeptide—as long as the epitope recognized by anantibody that specifically binds a phosphorylated GSK3β polypeptideresidue that includes a residue corresponding to phosphorylated Thr³⁹⁰residue of full-length, wild-type human GSK3β polypeptide, orphosphorylated Ser³⁸⁹ residue of full-length, wild-type, mouse GSK3βpolypeptide, is present.

Anti-Thr³⁹⁰-phosphorylated GSK3β antibodies or antigen-binding fragmentsthereof, or anti-Ser³⁸⁹-phosphorylated GSK3β antibodies orantigen-binding fragments thereof of the invention, can specificallybind Thr³⁹⁰-phosphorylated GSK3β polypeptide or Ser³⁸⁹-phosphorylatedGSK3β polypeptide with sub-nanomolar affinity. The binding affinitiescan be about 1×10⁻⁶, 1×10⁻⁷, 1×10⁻⁸, 1×10⁻⁹ or less, preferably about1×10⁻¹⁰ M or less, more preferably 1×10⁻¹¹ M or less. In certainembodiments the binding affinity is less than about 5×10⁻¹⁰M.

In some aspects of the invention, an antibody or antigen-bindingfragment thereof binds to a conformational epitope within thephosphorylated GSK3β polypeptide. To determine if the selectedanti-phosphorylated GSK3β antibodies bind to conformational epitopes,each antibody can be tested in assays using native protein (e.g.,non-denaturing immunoprecipitation, flow cytometric analysis of cellsurface binding) and denatured protein (e.g., Western blot,immunoprecipitation of denatured proteins). A comparison of the resultswill indicate whether the antibodies bind conformational epitopes. Insome embodiments antibodies that bind to native protein but notdenatured protein are those antibodies that bind conformationalepitopes,, and are preferred antibodies,

In some embodiments of the invention, antibodies competitively inhibitthe specific binding of a second antibody to its target epitope onphosphorylated GSK3β polypeptide. In some embodiments, the targetepitope comprises a phosphorylated residue that corresponds to Thr³⁹⁰ ofwild-type, full-length human GSK3β polypeptide or corresponds to Ser³⁸⁹of wild-type, full-length mouse GSK3β polypeptide. In some embodiments,the second antibody is phospo-S³⁸⁹ GSK3β. To determine competitiveinhibition, a variety of assays known to one of ordinary skill in theart can be employed. For example, competition assays can be used sodetermine if an antibody competitively inhibits binding tophosphorylated GSK3β (or Thr³⁹⁰-phosphorylated GSK3β orSer³⁸⁹-phosphorylated GSK3β) by another antibody (e.g., phospo-S³⁸⁹GSK3β). These methods may include cell-based methods employing flowcytometry or solid phase binding analysis. Other assays that evaluatethe ability of antibodies to cross-compete for phosphorylated GSK3βpolypeptide (or Thr³⁹⁰-phosphorylated GSK3β polypeptide orSer³⁸⁹-phosphorylated GSK3β) molecules in solid phase or in solutionphase, also can be used.

Certain antibodies competitively inhibit the specific binding of asecond antibody so its target epitope on phosphorylated GSK3βpolypeptide (or Thr³⁹⁰-phosphorylated GSK3β polypeptide orSer³⁸⁹-phosphorylated GSK3β by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 90% or 100%. Inhibition cars be assessed atvarious molar ratios or mass ratios; for example competitive bindingexperiments can be conducted with a 2-fold, 3-fold, 4-fold, 5-fold,7-fold, 10-fold or more molar excess of the first antibody over thesecond antibody.

Other antibodies of the invention may include antibodies thatspecifically bind to an epitope on phosphorylated GSK3β polypeptidedefined by a second antibody. To determine the epitope, one can usestandard epitope mapping methods known in the art. For example,fragments (polypeptides) of Thr³⁹⁰-phosphorylated GSK3β polypeptideantigen or Ser³⁸⁹-phosphorylated GSK3β polypeptide antigen that bind thesecond antibody can be used to determine whether a candidate antibodybinds the same epitope. In some embodiments, an epitope comprises aphosphorylated residue that corresponds to Thr³⁹⁰ of wild-type,full-length human GSK3β polypeptide. In some embodiments, an epitopecomprises a phosphorylated residue that corresponds to Ser³⁸⁹ ofwild-type, full-length mouse GSK3β polypeptide. In certain embodiments,the second antibody is phospho-S³⁸⁹ GSK3β antibody. For linear epitopes,overlapping polypeptides of a defined length (e.g., 5, 6, 7, 8 or moreamino acids) may be synthesized. The polypeptides preferably are offsetby 1 amino acid, such that a series of polypeptides covering every 4, 5,6, 7, or 8 amino acid fragment (respectively) of the phosphorylatedGSK3β polypeptide sequence are prepared. Fewer polypeptides can beprepared by using larger offsets, e.g., 2 or 3 amino acids. In addition,longer polypeptides (e.g., 9-, 10- or 11-mers) can be synthesized.Binding of polypeptides to antibodies can be determined using standardmethodologies including surface plasmon resonance (BIACORE) and ELISAassays. For examination of conformational epitopes, largerphosphorylated GSK3β polypeptide fragments, including in someembodiments Thr³⁹⁰-phosphorylated GSK3β polypeptide orSer³⁸⁹-phosphorylated GSK3β polypeptide, can be used. Other methods thatuse mass spectrometry to define conformational epitopes have beendescribed and can be used (see, e.g., Baerga-Ortiz et al., ProteinScience 11:1300-1308, 2002 and references cited therein). Still othermethods for epitope determination are provided in standard laboratoryreference works, such as Unit 6.8 (“Phage Display Selection and Analysisof B-cell Epitopes”) and Unit 9.8 (“identification of AntigenicDeterminants Using Synthetic Polypeptide Combinatorial Libraries”) ofCurrent Protocols in Immunology, Coligan et al., eds., John Wiley &Sons. Epitopes can be confirmed by introducing point mutations ordeletions into a known epitope, and then testing binding with one ormore antibodies to determine which mutations reduce binding of theantibodies.

An antibody or antigen-binding fragment thereof of the invention can belinked to a detectable label. A detectable label of the invention may beattached to antibodies or antigen-binding fragments thereof of theinvention by standard protocols known in the art. In some embodiments,the detectable labels may be covalently attached to ananti-phosphorylated GSK3β antibody or antigen-binding fragment thereofof the invention. The covalent binding can be achieved either by directcondensation of existing side chains or by the incorporation of externalbridging moieties. Many bivalent or polyvalent agents are useful incoupling protein molecules to other proteins, polypeptides or aminefunctions, etc. For example, the literature is replete with couplingagents such as carbodiimides, diisocyanates, glutaraldehyde, anddiazobenzenes. This list is not intended to he exhaustive of the variouscoupling agents known in the art but, rather, is exemplary of the morecommon coupling agents. Additional descriptions of detectable labelsuseful in the invention are provided elsewhere herein.

The invention, in part, also includes nucleic acid sequences that encodepolypeptide sequences for use in generating antibodies. For example, theinvention includes nucleic acid sequences that encode a phosphorylatedGSK3β polypeptide or fragment thereof described herein, and includes theuse of the nucleic acid sequences that may be used to producepolypeptides that can be used as antigens with which to raise antibodiesthat recognize phosphorylated GSK3β polypeptides described herein.

Additional nucleic acids of the invention include nucleic acids thatencode a GSK3β polypeptide, or an antibody or antigen-binding fragmentthereof of the invention. In certain embodiments, a nucleic acid of theinvention is a nucleic acid molecule that is highly homologous to anucleic acid that encodes a GSK3β polypeptide or an antibody orantigen-binding fragment thereof of the invention. Preferably thehomologous nucleic acid molecule comprises a nucleotide sequence that isat least about 90% identical to the nucleotide sequence that encodes theGSK3β polypeptide or antibody or antigen-binding fragment thereof. Morepreferably, the nucleotide sequence is at least about 95% identical, atleast about 97% identical, at least about 98% identical, or at leastabout 99% identical to a nucleotide sequence that encodes a GSK3βpolypeptide or an antibody or antigen-binding fragment thereof of theinvention. The homology can be calculated using various, publiclyavailable software tools well known to one of ordinary skill in the art.Exemplary tools include the BLAST system available from the website ofthe National Center for Biotechnology Information (NCBI) at the NationalInstitutes of Health. Similarly, the ammo acid sequence of a polypeptideuseful in methods and compositions of the invention may be at leastabout 90% identical to the amino acid sequence of a GSK3β polypeptide.The amino acid sequence may be at least about 95% identical, at leastabout 97% identical, at least about 98% identical, or at least about 99%identical to an amino acid sequence of a GSK3β polypeptide of theinvention.

One method of identifying highly homologous nucleotide sequences is vianucleic acid hybridization. Thus the invention also includes antibodieshaving phosphorylated GSK3β-binding properties (including but notlimited to Thr³⁹⁰-phosphorylated GSK3β polypeptide-binding properties orSer³⁸⁹-phosphorylated GSK3β polypeptide-binding properties) and otherfunctional properties described herein, and includes additional GSK3βpolypeptides that are encoded by nucleic acid molecules that hybridizeunder high stringency conditions to a nucleic acid that encodes anantibody or antigen-binding fragment thereof of the invention, or aGSK3β polypeptide of the invention, respectively. Identification ofrelated sequences can also be achieved using polymerase chain reaction(PCR) and other amplification techniques suitable for cloning relatednucleic acid sequences. Preferably, PCR primers are selected to amplifyportions of a nucleic acid sequence of interest, such as a CDR.

The term “high stringency conditions” as used herein refers toparameters with which the art is familiar. Nucleic acid hybridizationparameters may be found in references that compile such methods, e.g.,Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,Second Edition, Gold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989, or Current Protocols in Molecular Biology, P. M. Ausubel, etal., eds., John Wiley & Sons, Inc., New York. One example ofhigh-stringency conditions is hybridization at 65° C. in hybridizationbuffer (3.5× SSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02%Bovine Serum Albumin, 2.5 mM NaH₂PO₄(pH7), 0.5% SDS, 2 mM EDTA), SSC is0.15M sodium chloride/0.015M sodium citrate, pH 7; SDS is sodium dodecylsulphate; and EDTA is ethylenediaminetetracetic acid. Afterhybridization, a membrane upon which the nucleic acid is transferred iswashed, for example, in 2× SSC at room temperature and then at 0.1-0.5×SSC/0.1× SDS at temperatures up to 68° C.

Detectable Labels

Polypeptides and/or nucleic acids of the invention may be detectablylabeled for use in methods and/or compositions of the invention. A widevariety of detectable labels are available for use in methods of theinvention and may include labels that provide direct detection (e.g.,fluorescence, colorimetric, or optical, etc.) or indirect detection(e.g., enzyme-generated luminescence, epitope tag such as the FLAGepitope, enzyme tag such as horseradish peroxidase, labeled antibody,etc.). A variety of methods may be used to detect a detectable labeldepending on the nature of the label and other assay components. Labelsmay be directly detected through optical or electron density,radioactive emissions, nonradiative energy transfers, etc. or indirectlydetected with antibody conjugates, strepavidin-biotin conjugates, etc.Methods for using and detecting labels are well known to those ofordinary skill in the art. Methods of the invention may be used for invivo, in vitro, and/or ex vivo imaging, including but not limited toreal-time imaging. The presence of a labeled antibody in a subject canbe detected by in vivo, ex vivo, or in vitro imaging using standardmethods. Examples of detection methods include, but are not limited to,MRI, functional MRI, X-Ray detection, PET, CT imaging,immunohistochemistry. Western blot of tissues or cells, or by any othersuitable detection method.

The term “detestable label” as used here means a molecule preferablyselected from, but not limited to, fluorescent, enzyme, radioactive,metallic, biotin, chemiluminescent, and bioluminescent molecules. Asused herein, a detectable label may be a colorimetric label, e.g., achromophore molecule. In some aspects of the invention, a polypeptide oran antibody may be detectably labeled with a single or with two or moreof the detectable labels set forth herein, or other art-known detectablelabels.

Radioactive or isotopic labels may be, for example, ¹⁴C, ³H, ³⁵S, ¹²⁵I,and ³²P. Fluorescent labels may be any compound that emits anelectromagnetic radiation, preferably visible light, resulting from theabsorption of incident radiation and persisting as long as thestimulating radiation is continued.

Examples of fluorescent labels that may be used on polypeptides and/orantibodies of the invention and in methods of the invention include butare not limited to 2,4-dinitrophenyl, acridine, cascade blue, rhodamine,4-benzoylphenyl, 7-nitrobenz-2-oxa-1,3-diaxole,4,4-difluoro-4-bora-3a,4a-diaza-3-indacene and fluorescamine.Absorbance-based labels may be molecules that are detectable by thelevel of absorption of various electromagnetic radiation. Such moleculesmay be, for example, the fluorescent labels indicated above.

Chemiluminescent labels in this invention refer to compounds that emitlight as a result of a non-enzymatic chemical reaction. Methods of theinvention may also include the use of a luminescent detestablediagnostic molecule such as enhanced green fluorescent protein (EGFP),luciferase (Luc), or another detectable expression product.

Enzymatic methods for detection may be used including the use ofalkaline phosphatase and peroxidase. Additional enzymes may also be usedfor detection in methods and kits of the invention.

As used herein, fluorophores include, but are not limited toamine-reactive fluorophores that cover the entire visible andnear-infrared spectrum. Examples of such fluorophores include, but arenot limited to, 4-methylumbelliferyl phosphate, fluoresceinisothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC),BODIPY dyes; Oregon Green, rhodamine green dyes; the red-fluorescentRhodamine Red-X, Texas Red dyes; and the UV light-excitable CascadeBlue, Cascade Yellow, Marina Blue, Pacific Blue and AMCA-X fluorophores.Fluorophores may also include non-fluorescent dyes used in fluorescenceresonance energy transfer (FRET).

A labeled polypeptide or antibody of the invention can be prepared fromstandard moieties known in the art. As is recognized by one of ordinaryskill in the art, the labeling process for preparing a detectablelabeled polypeptide, antibody, or fragment thereof may vary according tothe molecular structure of the polypeptide or antibody and thedetectable label. Methods of labeling polypeptides and/or antibodieswith one or more types of detectable labels arc routinely used and arewell understood by those of ordinary skill in the art.

In some embodiments, it is contemplated that one may wish to firstderivative a polypeptide or antibody, and then attach the detectablelabel to the derivatized product. Suitable cross-linking agents for usein this manner include, for example, SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), and SMPT,4-succinimidyl-oxycarbonyl-methyl-(2-pyridyldithio)toluene. In someembodiments, a radionuclide may be coupled to a polypeptide, antibody,or antigen-binding fragment thereof by chelation.

Modulation of GSK3β Activity

Compositions (e.g., phosphorylated polypeptides, antibodies tophosphorylated GSK3β and derivatives/conjugates thereof, etc.) of thepresent invention have therapeutic utilities. For example, thesemolecules can be administered to cells in culture, e.g., in vitro or exvivo, or administered to a subject in vivo. In some embodiments thepolypeptides of the invention can be used to reduce activity of GSK3βactivity in a cell in some embodiments contacting a cell with a GSK3βpolypeptide comprising a phosphorylated residue that corresponds toThr³⁹⁰ in a full-length, wild-type human protein or Ser³⁸⁹ in afull-length wild-type mouse polypeptide, results in a decrease in theactivity of GSK3β. Polypeptides and compounds of the invention may becontacted with cells and/or administered to subjects in compositions.Compositions of the invention may include a carrier. In someembodiments, the carrier may be a pharmaceutically acceptable carrier.

Aspects of the invention relate to the discovery that GSK3β isphosphorylated at a C-terminal residue by p38/MAPK. As described hereinin the example section, p38/MAPK phosphorylates GSK3β at a C-terminalresidue that corresponds to Thr³⁹⁰ in a full-length, wild-type humanprotein or Ser³⁸⁹ in a full-length wild-type mouse polypeptide,resulting in a decrease in the activity of GSK3β. In some embodiments,methods of the invention relate to contacting a cell with a compoundthat phosphorylates GSK3β at a residue that corresponds to Thr³⁹⁰ in afull-length, wild-type human protein or Ser³⁸⁹ in a full-lengthwild-type mouse polypeptide. In order to regulate the activity of GSK3βin vitro or in vivo. In some embodiments a cell that is contacted with aGSK3β polypeptide has an endogenous GSK3β polypeptide present. Theendogenous GSK3β polypeptide may or may not be phosphorylated. In someembodiments, in order to reduce the activity of GSK3β, a cell iscontacted with both a phosphorylated GSK3β polypeptide comprising aphosphorylated residue corresponding to Thr³⁹⁰ in a full-length,wild-type human protein or Ser³⁸⁹ in a full-length wild-type mousepolypeptide, as well as a compound that results in phosphorylation ofGSK3β at a residue that corresponds to Thr³⁹⁰ in a full-length,wild-type human protein or Ser³⁸⁹ in a full-length wild-type mousepolypeptide.

In some embodiments the compound that results in phosphorylation ofGSK3β modulates the expression or activity of the p38 MAPK signalingpathway in the cell. p38 is a component of a mitogen-activated proteinkinase (MAPK) signaling cascade that responds to cellular conditionssuch as stress, cytokines, and/or growth factors. Upstream factors inthe p38/MAPK signaling pathway that respond to stimuli include, but arenot limited to, MAPKKK molecules including MLK3, TAK1, MEKK4 and ASK1.Downstream of these factors are the MAPKK molecules including MKK3/6.Downstream of these factors are the MAPK, molecules such as p38proteins, which influence further downstream factors such astranscription factors. It should be appreciated that a step or moleculein the p38 signaling pathway can be targeted in order to influence p38activity (e.g., MLK3, TAK1, MEKK4, ASK1, MKK3/6, p38α/β/γ/δ etc.). Insome non-limiting embodiments, activity of the p38 signaling pathway isupregulated through the use of inflammatory cytokines such as TNFalpha,IL-1 and IL-12, growth factors such as CSF-1, histamine, T cellreceptors, LPS and other TLR-ligands, or Dectin-ligands. In someembodiments, activity of the p38 signaling pathway is upregulatedthrough stress such as DNA damage. In certain embodiments DNA damage canbe caused by a procedure, examples of which include, but are not limitedto; exposure to UV, gamma-irradiation, X-ray, or chemotherapeutic drugs.Other non-limiting examples of sources of cellular stress includeoxidative stress, osmotic shock and heat, which may be non-limitingexamples of procedures that modulate phosphorylation; of GSK3β. Itshould be appreciated that methods of modulating the p38 signalingpathway are compatible with methods of the invention. In someembodiments of the invention, a compound that results in phosphorylationof GSK3β is p38.

In some embodiments, aspects of the invention relate to methods forincreasing the activity of GSK3β in a cell through reduction ofphosphorylation of a GSK3β residue that corresponds to Thr³⁹⁰ in afull-length, wild-type human protein or Ser³⁸⁹ in a full-lengthwild-type mouse polypeptide. In some embodiments a cell is contactedwith a compound that results in a decrease in levels of phosphorylationof GSK3β. In some embodiments the compound that results in a decrease inlevels of phosphorylation of GSK3β modulates expression or activity ofone or more components of the p38 MAPK signaling pathway. In someembodiments the compound that results in a decrease in levels ofphosphorylation of GSK3β inhibits p38 MAPK. It should be appreciatedthat a p38 MAPK inhibitor may inhibit expression (e.g., transcription,translation, and/or stability) of p38 MAPK and/or p38 MAPK activity. Aninhibitor may be a specific p38 MAPK inhibitor or a non-specificinhibitor (e.g., a non-specific kinase inhibitor) or a multi-targetinhibitor that inhibits p38 MAPK. An inhibitor may be a small molecule,an aptamer, an antibody, an RNAi, an antisense RNA, or any othersuitable molecule, or any combination thereof. In some embodiments, aprocedure such as one listed above herein may be used alone or inconjunction with a compound to modulate phosphorylation of GSK3β.

It should be appreciated that expression levels of any of the moleculesdiscussed herein, including GSK3β, may be detected using any suitabledirector indirect assay for detecting expression in a sample. In certainembodiments, protein expression may he detected by a Western blot. Insome embodiments a phospho-specific antibody is used, protein levels canalso be determined by an ELISA assay. In some embodiments the detectionof a modified or mutated form of a protein may be used to indicate theactivity level of a protein. For example the detection of aphosphorylation event on a specific residue of a protein may becorrelated with an increase or decrease in the activity of that proteinand in some embodiments an increase or decrease in the signalingactivity of a pathway involving that protein. In some embodiments,wherein the molecule is a kinase, kinase activity may be detected usinga kinase assay. In some embodiments activation of a protein can bemeasured by detection of levels of expression, activity,phosphorylation, etc., of a substrate of the molecule. It should beappreciated that an increase in activity of a molecule could represent achange in activity level of 1%, 2%, 5%, 10%, 20%, 25%, 30%, 50%, 75%,100%, 200%, up to 500%, and any value in between, relative to thewild-type activity level of the molecule. Similarly a decrease inactivity of a molecule could be a change in activity level of 1%, 2%,5%, 10%, 20%, 25%, 30%, 50%, 75%, up to 100%, and any value in between,relative to the wild-type activity level of the molecule.

Treatment

According so aspects of the invention, contacting a cell with aphosphorylated GSK3β polypeptide, and/or with a compound and/orprocedure that modulates GSK3β phosphorylation can occur in vivo or invitro. In some embodiments contacting a cell occurs in vivo and is amethod of treating a disease, disorder or condition that is associatedwith elevated or reduced GSK3β activity. As used herein “disorder”refers to any pathological condition associated with elevated or reducedGSK3β activity. In some embodiments the disorder or condition associatedwith elevated GSK3β activity is a neurological disorder or condition.Some non-limiting examples of neurological disorders or conditionsinclude stroke, cerebral ischemia, spinal cord trauma, head trauma,perinatal hypoxia, hypoglycemic neuronal damage, dementia (includingAIDS-induced dementia), Alzheimer's disease, Huntington's Chorea,amyotrophic lateral sclerosis, multiple sclerosis, ocular damage,cognitive disorders, idiopathic and drug-induced Parkinson's disease,amyotrophic lateral sclerosis, tremors, epilepsy, convulsions, migraine(including migraine headache), psychosis, schizophrenia, mood disorders(including depression, mania, bipolar disorders), trigeminal neuralgia,brain edema, pain (including acute and chronic pain states, severe pain,intractable pain, neuropathic pain, and post-traumatic pain), tardivedyskinesia, motor neuron disease, spinal muscular atrophy, progressivesupranuclear palsy, and multiple sclerosis. In some embodiments thedisease or condition associated with reduced GSK3β activity is cancer ordiabetes.

As used herein, the term treat, treated, or treating when used withrespect to a disorder refers to a prophylactic treatment that increasesthe resistance of a subject to development of the disease or, in otherwords, decreases the likelihood that the subject will develop thedisease as well as a treatment after the subject has developed thedisease in order to fight the disease or prevent the disease frombecoming worse. The term “treatment” embraces the prevention of adisorder or condition, and the inhibition and/or amelioration ofpre-existing disorders and conditions. A subject may receive treatmentbecause the subject has been determined to be as risk of developing adisorder or condition, or alternatively, the subject may have such adisorder or condition. Thus, a treatment may prevent, reduce oreliminate a disorder or condition altogether or prevent it from becomingworse.

As used herein, the term “subject” refers to a human or non-human mammalor animal non-human mammals include livestock animals, companionanimals, laboratory animals, and non-human primates. Non-human subjectsalso specifically include, without limitation, chickens, horses, cows,pigs, goats, dogs, cats, guinea pigs, hamsters, mink, and rabbits. Insome embodiments of the invention, a subject is a patient. As usedherein, a “patient” refers to a subject who is under the care of aphysician or other health care worker, including someone who hasconsulted with, received advice from or received a prescription or otherrecommendation from a physician or other health care worker.

As used herein, the term “cancer” refers to an uncontrolled growth ofsells that may interfere with the normal functioning of the bodilyorgans and systems, and includes both . primary and metastatic tumors.Primary tumors or cancers that migrate from their original location andseed vital organs can eventually lead to the death of the subjectthrough the functional deterioration of the affected organs. Ametastasis is a cancer cell or group of cancer cells, distinct from theprimary tumor location, resulting from the dissemination of cancer cellsfrom the primary tumor to other parts of the body. Metastases mayeventually result in death of a subject.

As used herein, the term “cancer” includes, but is not limited to, thefollowing types of cancer: breast cancer (including carcinoma in situ),biliary tract cancer; bladder cancer; brain cancer includingglioblastomas and medulloblastomas; cervical cancer; choriocarcinoma;colon cancer; endometrial cancer; esophageal cancer; gastric cancer;hematological neoplasms including acute lymphocytic and myelogenousleukemia; T-cell acute lymphoblastic leukemia/lymphoma; hairy cellleukemia; chromic myelogenous leukemia, multiple myeloma;AIDS-associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms including Bowen's disease and Paget's disease;liver cancer; lung cancer; lymphomas including Hodgkin's disease andlymphocytic lymphomas; mesothelioma, neuroblastomas; oral cancerincluding squamous cell carcinoma; ovarian cancer including thosearising from epithelial cells, stromal cells, germ cells and mesenchymalcells; pancreatic cancer; prostate saucer; rectal cancer; sarcomasincluding leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma,and osteosarcoma; skin cancer including melanoma, Merkel cell carcinoma,Kaposi's sarcoma, basal cell carcinoma, and squamous cell cancer;testicular cancer including germinal tumors such as seminoma,non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germcell tumors; thyroid cancer including thyroid adenocarcinoma; andmedullar carcinoma; and renal cancer including adenocarcinoma and Wilmstumor. Non-limiting examples of precancerous conditions includedysplasia, premalignant lesions, adenomatous colon polyp, and carcinomain-situ such as Ductal carcinoma in-situ (DCIS), etc. Other cancers thatcan be treated with methods of the invention will be known to those ofordinary skill in the art. In some embodiments of the invention, thecancer is melanoma. In certain embodiments the cancer is adenocarcinoma,in some embodiments the cancer is a solid tumor cancer. A cancer thatmay be treated or assayed using methods of the invention also mayinclude breast cancer, lung cancer, prostate cancer, mesothelioma, etc.

Selecting Treatment

Described herein are methods for assessing effectiveness of acomposition such as a phosphorylated GSK3β polypeptide or a compoundthat modulates the level of phosphorylation of GSK3β for treatment of adisease or condition, in some embodiments a cell will be contacted witha phosphorylated GSK3β polypeptide and/or a compound that modulates thelevel of phosphorylation of GSK3β, and the effect of this treatment onthe cell will be monitored. In some embodiments the viability of thecell (e.g., a neuronal cell or a cancer cell) will be monitored and willbe indicative of the effectiveness of the treatment. In someembodiments, the cell is taken from a subject who has a neurologicaldisease or disorder or a subject who has cancer, in certain embodiments,sample sources for the cell may include tissues, including but notlimited to, lymph tissues; body fluids (e.g., blood, lymph fluid, etc.),cultured cells, cell lines; histological slides; tissue embedded inparaffin; etc. The term “tissue” as used herein refers to both localizedand disseminated cell populations including, but not limited tot brain,heart, serum, breast, colon, bladder, epidermis, skin, uterus, prostate,stomach, testis, ovary, pancreas, pituitary gland, adrenal gland,thyroid gland, salivary gland, mammary gland, kidney, liver, intestine,spleen, thymus, bone marrow, trachea, and lung. Invasive andnon-invasive techniques can be used to obtain such samples and are welldocumented in the art. A control cell sample may include a cell, atissue, or may be a lysate of either. In some embodiments, a controlsample may be a sample that is a cell or from a subject that is free ofa neurological disease or disorder or cancer and/or free of aprecancerous condition. In some embodiments, a control sample may be asample that is a cell or from a subject that has a neurological diseaseor disorder or cancer or a precancerous condition. It will be understoodthat controls according to the invention may be, in addition topredetermined values, samples of materials tested in parallel with theexperimental materials. Examples include samples from controlpopulations or control samples generated through manufacture to hetested in parallel with the experimental samples.

In some embodiments, factors such as the level of cell growth,proliferation, and/or viability of cells in a sample may he measured. Insome embodiments, measurements of cell growth or proliferation can becorrelated to levels of cell viability, whereas in other embodiments,cell viability may be measured directly. These factors can be determinedin a number of ways when carrying out the various methods of theinvention. In one measurement, the level of cell growth, proliferation,or viability of cells in a test sample is measured in relation to acontrol sample. In some embodiments, a control sample and a test samplemay be taken from the sense subject. The test sample may be treated witha composition comprising a phosphorylated GSK3β polypeptide and/or acompound that modulates phosphorylation of GSK3β. In some embodimentsthe control sample may be treated with either a composition comprising aphosphorylated GSK3β polypeptide or a compound that modulatesphosphorylation of GSK3β, or with neither of these. The control and testsamples may then be compared for the levels of such characteristics ascell growth, cell proliferation, and/or cell viability of cells in thesample, using art-known methods.

In some embodiments the cell is from a subject who has a neurologicaldisease or disorder and the treatment is intended to increase cellsurvival. In this embodiment if the test sample shows increased cellgrowth, and/or proliferation and/or viability, relative to the controlsample, then this would be interpreted to mean that cells in the testsample respond to treatment with a phosphorylated GSK3β polypeptideand/or a compound that modulates phosphorylation of GSK3β.

In some embodiments the cell is from a subject who has cancer and thetreatment with a compound that modulates phosphorylation of GSK3β isintended to reduce cell survival. In this embodiment if the test sampleshows decreased cell growth, and/or proliferation and/or viability,relative to the control sample, then this would be interpreted to meanthat cells in the test sample, respond to treatment with a compound thatmodulates phosphorylation of GSK3β.

In some embodiments, a test sample and a control sample may includecells from different disorders. In some embodiments, a control samplemay be a cell from a type of disorder that is known to respond totreatment with a phosphorylated GSK3β polypeptide and/or a compound thatmodulates phosphorylation of GSK3β. In such embodiments, if the testsample responds similarly to the control sample, then the test samplewould be interpreted as responding to treatment. In other embodiments, acontrol sample may be a cell from a type of disorder that is known notto respond so treatment with a phosphorylated GSK3β polypeptide and/or acompound that modulates phosphorylation of GSK3β. In such embodiments,if the test sample responds similarly so the control sample then thetest sample would be interpreted as not responding to treatment. It willbe understood that the interpretation of a comparison between a testsample and a control sample will depend on the nature of both samples.

One possible measurement of the level of cell growth, proliferation,and/or viability of cells in a sample is a measurement of absolutelevels of cell growth, proliferation, and/or viability. Anothermeasurement of the level of cell growth, proliferation, or viability ofcells in a sample is a measurement of the change in the level of cellgrowth, proliferation, or viability of cells over time. This may beexpressed in an absolute amount or may be expressed in terms of apercentage increase or decrease over time. Methods and assays of theInvention may be combined with other methods and assays in determiningthe level of cell growth, proliferation, and/or viability of cells in asample, and in determining an optimal treatment strategy for a patient.

In some embodiments, a control value may be a predetermined value, whichcan take a variety of forms. It can be a single cut-off value, such as amedian or mean. It can be established based upon comparative groups,such as in groups having normal amounts of cell growth, proliferation,and/or viability, and groups having abnormal amounts of cell growth,viability, and/or proliferation. For example, in some embodiments acontrol sample that is taken from a subject with a disorder and is nottreated with a phosphorylated GSK3β polypeptide and/or a compound thatmodulates phosphorylation of GSK3β may be considered to have normallevels of cell growth, viability, and proliferation tor a cancer cell.In such embodiments, a test sample that is taken from the same subjectand treated with a phosphorylated GSK3β polypeptide and/or a compoundthat modulates phosphorylation of GSK3β may be considered to haveabnormal levels of cell growth, viability, and/or proliferation.

In another embodiment, a cell from a subject who does not have adisorder may be considered to have normal levels of cell growth,viability, and proliferation. In this embodiment, a cell taken from asubject who has a disorder, and not treated with a phosphorylated GSK3βpolypeptide and/or a compound that modulates phosphorylation, may beconsidered to have abnormal levels of cell growth, viability, and/orproliferation, whereas a cell taken from the same subject and treatedwith a phosphorylated GSK3β polypeptide and/or a compound that modulatesphosphorylation .may be found to have levels of cell growth, viability,and/or proliferation that approach the normal level, which in suchembodiments, would be the levels for a cell from a subject who does nothave a disorder.

Based at least in part on results of hi vitro methods discussed herein,a predetermined value can be arranged. For example, test samples and thesubjects from which the samples were extracted, are divided equally (orunequally) into groups, such as a low-response group, a medium-responsegroup and a high-response group, where response refers to response ofthe sample from each group to treatment with a phosphorylated GSK3βpolypeptide and/or a compound that modulates phosphorylation usingmethods described herein. Test samples and subjects may be divided intoquadrants or quintiles, the lowest quadrant or quintile beingindividuals with the lowest response and the highest quadrant orquintile being individuals with She highest response. Individuals withthe highest level of response to the treatment would be considered themost likely to respond to the treatment. However individuals in low andmedium response groups may also be found to respond to the treatment.

The predetermined value, of course, will depend upon the particularpopulation selected. For example, an apparently healthy population willhave a different ‘normal’ range than will a population that is known tohave a disorder. In addition, values may be different for differentdisorders, or for different populations or individuals. Accordingly, thepredetermined value selected may take into account the category in whichan individual or cell falls. Appropriate ranges and categories can beselected with no more than routine experimentation by those of ordinaryskill in the art. As used herein, “abnormal” means not normal ascompared to a control. By abnormally high or low it is meant high or lowrelative to a selected control.

It is also possible to use measurements of cell growth, proliferation,and/or viability to monitor changes in the levels of these factors overtime in a cell sample. For example, in some embodiments it is expectedthat treatment of a cancer cell with a compound that modulates GSK3βphosphorylation will lead to a decrease in levels of cell growth,proliferation, and/or viability relative to a control sample of a cancercell that is not treated with a phosphorylated GSK3β polypeptide and/ora compound that modulates GSK3β phosphorylation. In some embodiments itis expected that treatment of a neuronal cell with a phosphorylatedGSK3β polypeptide and/or a compound that modulates GSK3β phosphorylationwill lead to an increase in levels of cell growth, proliferation, and/orviability relative to a control sample of a neuronal cell that is nottreated with a phosphorylated GSK3β polypeptide and/or a compound thatmodulates GSK3β phosphorylation. Accordingly, one can monitor levels ofcell growth, proliferation, and/or viability over time so determine ifthere is a change in the levels of these factors in a subject or in acell culture. In some embodiments, changes in levels of cell growth,proliferation, and/or viability greater than 0.1% may be considered toindicate effectiveness of the treatment on the levels of these factors.In some embodiments, the reduction in levels of cell growth, viabilityand/or proliferation, which indicate effectiveness of the treatment onthese factors, is a redaction greater than 0.2%, greater than 0.5%,greater than 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%, including eachpercentage in between these values. Increases or decreases in the levelsof cell growth, proliferation, and/or viability of cells over time mayindicate a change in responsiveness to treatment in a sample or subject.To make a determination of a change in responsiveness to treatment in asubject over time, multiple samples may be obtained from the subject atdifferent times and the samples tested for levels of cell growth,proliferation, and/or viability of cells. Resulting values may becompared to each other as a measure of change over time.

Methods of selecting a treatment may be useful to assess and/or adjusttreatment of subjects already receiving a drug or therapy (e.g.,radiation treatment or surgery) for treating a disorder or condition.Based on the determination of the response of a cell to administrationof s phosphorylated GSK3β polypeptide and/or a compound that modulatesGSK3β phosphorylation, it may be appropriate to alter a therapeuticregimen for s subject. For example, determination that a cell respondsto administration of a phosphorylated GSK3β polypeptide and/or acompound that modulates GSK3β phosphorylation in a subject who hasreceived or is receiving a treatment for a neurological disorder, canceror precancerous-condition treatment may indicate that the treatmentregimen should be adjusted (e.g., the dose or frequency of dosing,increased, new treatment initiated, etc.). For example, a reduction incancer cell viability after contact with a compound that modulates GSK3βphosphorylation indicates that the cancer is responsive to the treatmentand that the treatment may be useful to treat that cancer. Similarly, anincrease in neuronal cell viability in a subject who has a neurologicaldisorder after contact with a GSK3β polypeptide and/or a compound thatmodulates GSK3β phosphorylation indicates that the neurological disorderis responsive to the treatment and that the treatment may be useful totreat that neurological disorder. Different parameters can be assessedto determine appropriate optimized treatment regimens for a givenpatient's disorder.

Administration

According to aspects of the invention, polypeptides, compounds, andcompositions are administered in effective amounts. In a subject who hasa disorder that is associated with elevated GSK3β activity, an effectiveamount of a composition is the amount effective to decrease the level ofGSK3β activity in the subject. In a subject who has a disorder that isassociated with reduced GSK3β activity, an effective amount of acomposition is the amount effective to increase the level of GSK3βactivity in the subject. Phosphorylated GSK3β polypeptides associatedwith the invention and/or compositions that increase phosphorylation ofGSK3β may be administered in effective amounts to prevent and/or treatdisorders or conditions that are associated with elevated GSK3β activitysuch as neurological disorders or conditions. Compositions that resultin decreased phosphorylation of GSK3β may he administered in effectiveamounts to prevent and/or treat disorders or conditions that areassociated with reduced GSK3β activity such as cancer. Typically aneffective amount of a composition for treatment of a disorder orcondition will be determined in clinical trials, establishing aneffective dose for a test population versus a control population in ablind study. In some embodiments, an effective amount will be an amountthat results in a desired response, e.g.. an amount that diminishes oreliminates phosphorylation and/or activity of GSK3β. Thus, an effectiveamount may be the amount that when administered increases or decreasesthe phosphorylation and/or activity of GSK3β from the amount that wouldoccur in the subject or tissue without the administration of thecomposition of the invention. In the case of treating s particulardisease or condition the desired response is inhibiting the progressionof the disease or condition. This may involve only slowing theprogression of the disease temporarily, although more preferably, itinvolves halting the progression of the disease permanently. This can bemonitored by routine diagnostic methods known to one of ordinary skillin the art for any particular disease. The desired response to treatmentof the disease or condition also can be delaying the onset or evenpreventing the onset of the disease or condition.

Effective amounts of therapeutic compounds such as an effective amountof a phosphorylated GSK3β polypeptide and/or an effective amount of acomposition that modulates GSK3β phosphorylation may also be determinedby assessing physiological effects of administration on a cell orsubject, such as a decrease of disease symptoms followingadministration. Other assays will be known to one of ordinary skill inthe art and can be employed for measuring the level of the response to atreatment. The amount of a treatment may be varied for example, byincreasing or decreasing the amount of a therapeutic composition, bychanging the therapeutic composition administered, by changing the routeof administration, by changing the dosage timing and so on. Theeffective amount will vary with the particular condition being treated,the age and physical condition of the subject being treated, theseverity of the condition, the duration of the treatment, the nature ofthe concurrent therapy (if any), the specific route of administration,and the like factors within the knowledge and expertise of the healthpractitioner. For example, an effective amount may depend upon thedegree to which an individual has abnormally low or high levels ofphosphorylation of GSK3β polypeptide.

Effective amounts will also depend, of course, on the particularcondition being treated, the severity of the condition, the individualpatient parameters including age, physical condition, size and weight,the duration of the treatment, the nature of concurrent therapy (ifany), the specific route of administration and like factors within theknowledge and expertise of the health practitioner. These factors arewell known to those of ordinary skill in the art and can be addressedwith no more than routine experimentation. It is generally preferredthat a maximum dose of a composition according to the invention (aloneor in combination with other therapeutic agents) be used, that is, thehighest sale dose according to sound medical judgment. It will beunderstood by those of ordinary skill in the art, however, that apatient may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.

A pharmaceutical compound or composition dosage may be adjusted by theindividual physician or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount typically varies from0.01 mg/kg so about 1000 mg/kg, preferably from about 0.5 mg/kg to about200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg,in one or more dose administrations daily, for one or more days.

The absolute amount will depend upon a variety of factors, including thematerial selected for administration, whether the administration is insingle or multiple doses, and individual subject parameters includingage, physical condition, size, weight, and the stage of the disease orcondition. These factors are well known to those of ordinary skill inthe art and can be addressed with no more than routine experimentation.

Pharmaceutical compounds or compositions of the invention may beadministered alone, in combination with each other, and/or incombination with other drug therapies, or other treatment regimens thatare administered to subjects with disorders associated with theinvention.

A pharmaceutical compositions used in the foregoing methods preferablyare sterile and contain an effective amount of a therapeutic compoundthat will modulate the level of GSK3β activity for a level that producesthe desired response in a unit of weight or volume suitable foradministration to a patient.

The doses of pharmaceutical compounds or compositions of the inventionadministered to a subject can be chosen in accordance with differentparameters, in particular in accordance with the mode of administrationused and the state of the subject. Other factors include the desiredperiod of treatment. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localised delivery route) may beemployed to the extent that patient tolerance permits.

Various modes of administration will be known to one of ordinary skillin the art which effectively deliver a pharmaceutical composition of theinvention (e.g., a phosphorylated GSK3β polypeptide and/or a compositionthat modulates phosphorylation of GSK3β) to a desired tissue, cell orbodily fluid. Methods for administering a pharmaceutical compound orcomposition of the invention may be topical, intravenous, oral,intracavity, intrathecal, intrasynovsal, buccal, sublingual, intranasal,transdermal, intravitreal, subcutaneous, intramuscular and intradermaladministration. The invention is not limited by the particular modes ofadministration disclosed herein. Standard references in the art (e.g.,Remington's Pharmaceutical Sciences, 18th edition, 1990) provide modesof administration and formulations tor delivery of variouspharmaceutical preparations and formulations in pharmaceutical carriers.Other protocols which are useful for the administration of a compound orcomposition of the invention will be known to one of ordinary skill inthe art, in which the dose amount, schedule of administration, sites ofadministration, mode of administration (e.g., intra-organ) and the likevary from those presented herein.

Administration of a phosphorylated GSK3β polypeptide, or otherpharmaceutical compound or composition of the invention to mammals otherthan humans, e.g., for testing purposes or veterinary therapeuticpurposes, is carried out under substantially the same conditions asdescribed above. It will be understood by one of ordinary skill in theart that this invention is applicable to both human and animal diseaseswhich can be treated by a phosphorylated GSK3β polypeptide, or otherpharmaceutical compound of the invention.

When administered, the pharmaceutical preparations of the invention axeapplied in pharmaceutically-acceptable amounts and inpharmaceutically-acceptable compositions. The term “pharmaceuticallyacceptable” means a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients. Suchpreparations may routinely contain salts, buffering agents,preservatives, compatible carriers, and optionally other therapeuticagents. When used in medicine, the salts should be pharmaceuticallyacceptable, but non-pharmaceutically acceptable salts may convenientlybe used to prepare pharmaceutically-acceptable salts thereof and are notexcluded from the scope of the invention. Such pharmacologically andpharmaceutically-acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic,succinic, and the like. Also, pharmaceutically-acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts. Preferred components of the composition aredescribed above in conjunction with the description of the GSK3βpolypeptides and compositions of the invention that modulatephosphorylation of GSK3β.

A phosphorylated GSK3β polypeptide, or other therapeutic compound of theinvention may be combined, if desired, with apharmaceutically-acceptable carrier. The term“pharmaceutically-acceptable carrier” as used herein means one or morecompatible solid or liquid fillers, diluents or encapsulating substanceswhich are suitable for administration into a human. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the phosphorylated GSK3β polypeptide, or othertherapeutic compound of the invention, and with each other, in a mannersuch that there is no interaction which would substantially impair thedesired pharmaceutical efficacy.

A pharmaceutical composition of the invention may contain suitablebuffering agents, as described above, including: acetate, phosphate,citrate, glycine, borate, carbonate, bicarbonate, hydroxide (and otherbases) and pharmaceutically acceptable salts of the foregoing compounds.

A pharmaceutical composition of the invention, also may contain,optionally, suitable preservatives, such as: benzalkonium chloride;chlorobutanol; parabens and thimerosal. The pharmaceutical compositionsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well-known in the art of pharmacy. All methodsinclude the step of bringing the active agent into association with acarrier which constitutes one or more accessory ingredients. In general,the compositions are prepared by uniformly and intimately bringing theactive compound into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration may comprise aphosphorylated GSK3β polypeptide, or other therapeutic compound of theinvention. This preparation may be formulated according to known methodsusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation also may be a sterile injectable solutionor suspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water. Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono-ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Carrier formulation suitable fororal, subcutaneous, intravenous, intramuscular, etc. administrations canbe found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

Screening

GSK3β polypeptides comprising a phosphorylated residue that correspondsto residue Thr³⁹⁰ in a full-length, wild-type, human GSK3β amino acidsequence, or that corresponds to residue Ser³⁸⁹ in a full-length,wild-type, mouse GSK3β amino acid sequence of the invention may also beuseful in methods of screening for candidate agents that modulate levelsof phosphorylated GSK3β polypeptides in cells, tissues, and/or subjects.Methods can include contacting a GSK3β polypeptide comprising aphosphorylated residue that corresponds to residue Thr³⁹⁰ in afull-length, wild-type, human GSK3β amino acid sequence, or thatcorresponds to residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3βamino acid sequence, with p38 MAPK and a putative modulating compoundunder suitable conditions for phosphorylation of the residue thatcorresponds to residue Thr³⁹⁰ in a full-length, wild-type, human GSK3βamino acid sequence, or that corresponds to residue Ser³⁸⁹ in afull-length, wild-type, mouse GSK3β amino acid sequence. Levels ofphosphorylation of the Thr³⁹⁰ or Ser³⁸⁹ residue in the contactedpolypeptide would then be determined and compared to a controlpolypeptide that is not contacted with the putative modulating compound.An increase in the level of phosphorylation of the GSK3β polypeptide inthe contacted polypeptide relative to the control is indicative of acompound capable of increasing the level of phosphorylation of the GSK3βpolypeptide. An increase in the phosphorylation of the GSK3β polypeptidein a subject known to have a neurological disorder is indicative thatthe candidate agent/compound is capable of decreasing the level of GSK3βactivity and may be useful to reduce and/or eliminate a neurologicalcondition in cells, tissues, and/or subjects. A decrease in thephosphorylation of the GSK3β polypeptide in a subject known to have acancer or precancerous condition is indicative that the candidateagent/compound is capable of increasing the level of GSK3β activity andmay be useful to reduce and/or eliminate a cancer or precancerouscondition in cells, tissues, and/or subjects.

The assay mixture comprises a putative modulating compound. The putativemodulating compound is preferably an antibody, a small organic compound,or a polypeptide, and accordingly can be selected from combinatorialantibody libraries, combinatorial protein libraries, or small organicmolecule libraries. Typically, a plurality of reaction mixtures are runin parallel with different compound concentrations to obtain a differentresponse to the various concentrations. Typically, one of theseconcentrations serves as a negative control, i.e., at zero concentrationof compound or at a concentration of compound below the limits of assaydetection.

Candidate compounds encompass numerous chemical classes, althoughtypically they are organic compounds, proteins or antibodies (andfragments thereof that bind antigen). In some preferred embodiments, thecandidate compounds are small organic compounds, i.e., those having amolecular weight of more than 50 yet less than about 2500, preferablyless than about 1000 and, more preferably, less than about 500.Candidate compounds comprise functional chemical groups necessary forstructural interactions with polypeptides and/or nucleic acids, andtypically include at least an amine, carbonyl, hydroxyl, or carboxylgroup, preferably at least two of the functional chemical groups andmore preferably at least three of the functional chemical groups. Thecandidate compounds can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate compounds also canbe biomolecules such as polypeptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like.

Candidate compounds are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds, for example,numerous means are available tor random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides, synthetic organic combinatorial libraries,phage display libraries of random or non-random polypeptides,combinatorial libraries of proteins or antibodies, and the like.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are available or readily produced.Additionally, natural and synthetically produced libraries and compoundscan be readily be modified through conventional chemical, physical, andbiochemical means. Further, known agents may be subjected to directed orrandom chemical modifications such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs of theagents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc., which may be used to facilitate optimalprotein-protein and/or protein-agent binding. Such a reagent may alsoreduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The order of addition of components, incubation temperature, time ofincubation, and other parameters of the assay may be readily determined.Such experimentation merely involves optimization of the assayparameters, not the fundamental composition of the assay, incubationtemperatures typically are between 4° C. and 40° C. Incubation timespreferably are minimized to facilitate rapid, high throughput screening,and typically are between 0.1 and 10 hours. After incubation, thepresence or absence of and/or the level of phosphorylation of GSK3βpolypeptides described herein is detected by any convenient methodavailable to the user. For example, the level of phosphorylation ofGSK3β. polypeptides can be determined through the measure of adetectable label using standard methods and as described herein.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

Kits

Also within the scope of the invention are kits that may includepolypeptides, compounds, antibodies, and/or compositions of theinvention and instructions for use. The kits can further contain atleast one additional reagent, such as one or more additionalpolypeptides of the invention. In some embodiments kits of the inventionmay he useful for determining a treatment regimen for a disorder orcondition such as a neurological disorder or a cancer. An example ofsuch a kit may include a composition including one or morephosphorylated GSK3β polypeptide and/or one or more compounds formodulating phosphorylation of GSK3β, and instructions for use of thepolypeptides and/or compounds tot determining whether the compositionscan be used as a treatment regimen for a disorder such as a neurologicaldisorder or a cancer. Kits of the invention may also be useful fortreating a disorder such as a neurological disorder or a cancer. Anexample of such a kit may include one or more phosphorylated GSK3βpolypeptides and/or one or more compounds for modulating phosphorylationof GSK3β, and instructions for use of a combination of the polypeptidesand compounds for treating the disorder.

Kits containing polypeptides and/or compounds tor modulatingphosphorylation of GSK3β of the invention can be prepared for contactinga cell in vitro or in vivo. The components of the kits can be packagedeither in aqueous medium or in lyophilized form.

A kit may comprise a carrier being compartmentalized to receive in closeconfinement therein one or more container means or series of containermeans such as test tubes, vials, flasks, bottles, syringes, or the like.A first of said container means or series of container means may containone or more phosphorylated GSK3β polypeptides and/or one or morecompounds for phosphorylation of GSK3β.

A kit of the invention can include a description of use of Shepolypeptides, antibodies, compounds and/or compositions forparticipation in any biological or chemical mechanism disclosed herein.Kits can further include a description of activity of the condition intreating the pathology, as opposed to the symptoms of the condition.That is, a kit can include a description of use of the polypeptides,antibodies, compounds, and/or compositions as discussed herein. A kitalso can include instructions for use of a combination of two or morecompositions of the invention, or instruction for use of a combinationof a composition of the Invention and one or more other compoundsindicated for determining a treatment regimen for a disorder.Instructions also may be provided for administering the composition byany suitable technique as previously described.

The kits described herein may also contain one or more containers, whichmay contain a composition and other ingredients as previously described.The kits also may contain instructions for mixing, diluting, and/oradministering or applying the compositions of the invention in somecases. The kits also can include other containers with one or moresolvents, surfactants, preservative and/or diluents (e.g., normal saline(0.9% NaCl), or 5% dextrose) as well as containers for mixing, dilutingor administering the components in a sample or to a subject in need ofsuch treatment.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the polypeptide,compound and/or composition provided is a dry powder, is may bereconstituted by She addition of a suitable solvent, which may also beprovided. In embodiments where liquid forms of the composition are used,the liquid form may be concentrated or ready to use. The solvent willdepend on the composition and the mode of use or administration.Suitable solvents for drug compositions are well known, for example aspreviously described, and are available in the literature. The solventwill depend on the composition and the mode of use or administration.

An example of a kit useful according to the invention is shown in FIG.16. The kit (10) shown in FIG. 16 includes a set of containers forhousing compounds such as a phosphorylated GSK3β polypeptide (12) andother compounds (14) as well as instructions (20).

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including: literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein by reference.

EXAMPLES Example 1

Phosphorylation by p38 MAP Kinase as an Alternative Pathway for GSK3βinactivation

The p38 MAPK is activated through phosphorylation primarily by MAPKKinase (MKK)3 and MKK6 in response to cellular stress and cytokines. Thep38 MAPK pathway functions in the control of differentiation, blockadeof proliferation, and in induction of apoptosis (l). It is alsoactivated in response to DNA double stranded breaks (DSBs) induced byionizing irradiation or chemotherapeutic drugs, and participates in theinduction of a G2/M cell cycle checkpoint (2, 3). p38 MAPK can alsopromote survival (4-6) by unknown mechanisms. During T cell receptor β(TCRβ) rearrangement, V(D)J-mediated DSBs also activate p38 MAPK inimmature thymocytes at the double negative 3 (DN3) stage of development(7, 8). Expression of a constitutively active mutant of MKK6 [MKK6(Glu)]in thymocytes of transgenic mice (MKK6 transgenic mice) activates ap53-mediated G2/M phase cell cycle checkpoint (8). Likerecombination-activating gene (Rag) deficiency, persistent activation ofp38 MAPK. interferes with differentiation of thymocytes beyond the DN3stage. However, MKK6 transgenic thymocytes but not Rag^(−/−) thymocytessurvive and accumulate in vivo (8), suggesting that p38 MAPK may alsoprovide a survival signal. Gene expression profile analysis comparingRag^(−/−) and MKK6 DN3 thymocytes revealed that the MKK6 DN3 thymocytesexpressed more c-myc and lef (FIG. 5), two transcription factorsassociated with cell survival (9-11).

Mice

The MKK6(Glu) transgenic mice have been previously described (7, 8).Wildtype and Rag^(−/−) mice were purchased from Jackson Laboratory (BarHarbor, Me.). Procedures that involved mice were approved byinstitutional guidelines for animal care.

Plasmids

pGEX-GST-GSK3β and pGEX-GST-GSK3β T⁴³A both with the K⁸⁴A kinaseinactivating mutation were gifts from Dr. Mien-Chie Hung. Site directedmutagensis using the Transformer kit (Clontech, Mountain View, Calif.)was used to generate kinase inactive pGEX-GST-GSK3β-T³⁹⁰A andpGEX-GST-GSK3β-T³⁹⁰A /T⁴³A double mutants for use as substrates. Thesame site directed mutagensis technique was used to reverse the K⁸⁴Amutation to generate kinase active pGEX-GST-GSK3β wildtype and mutantconstructs for their use in GSK3β in vitro kinase assays. Wildtype humanGSK3β was subcloned into the expression vector pEGZ-HA, a gift fromIngolf Berberich (University of Wurzburg, Wursburg, Germany). Expressionplasmids for wildtype p38 MAPK and constitutively active MKK6 (24) werealso used. Mouse GSK-3β with a C-terminal 3XFLAG tag was subcloned intothe mammalian expression vector pEFS/FRT/V5/D-TOPO (Invitrogen,Carlsbad, Calif.). The point mutation at S³⁸⁹ (Ala) was introduced usingthe QuikchangeII XL mutagenesis kit (Stratagene, La Jolla, Calif.).

Cell Cultures

Thymocytes were isolated from wildtype, Rag^(−/−) or MKK6(Glu)transgenic mice. 293T cells were transiently transfected with theindicated expression constructs using calcium phosphate. When specified,293T cells were treated with SB203580 (5 μM) and Wortmanin (1 μM)(Calbiochem, San Diego, Calif.). Wildtype, GSK3α^(−/−) and GSK3β^(−/−)ES cells (17), as well as WT and MKK3^(−/−)MKK6^(−/−) MEF (22) have beenpreviously described, and were also treated with SB203580 as describedabove for the indicated periods of time. For in vivo inhibition of p38MAPK, wildtype mice were intraperitoneally (i.p.) injected with SB203580or vehicle alone, and after 18 hours brain and thymocytes were harvestedto prepare whole cell lysates.

Reverse Transcription-Polymerase Chain Reaction

Total RNA was extracted using RNAeasy mini kit (Qiagen, Valencia,Calif.) and transcribed into cDNA using oligo(dT) primers and reversetranscriptase (Invitrogen, Carlsbad, Calif.) that was used to examineβ-catenin expression by semiquantitative RT-PCR. The primers forβ-catenin were (5′ACAGCACCTTCAGCACTCT3′ (SEQ ID NO:91) and5′AAGTTCTTGGCTATTACGACA) (SEQ ID NO:92) and the primers for Actin were5′GTGGGGGCGCCCCAGCGCACCA3′ (SEQ ID NO:93) and 5′CTT CCT TAA TGT CAC GCACGA TTT C 3′ (SEQ ID NO:94)

Western Blots and Immunoprecipitations

Whole cell extracts were prepared in Triton lysis buffer (TLB) aspreviously described (25, 26). Nuclear extracts were made from cells aspreviously described (27). Immunoprecipitations and Western blotting wasperformed as described in (8). Anti-Akt, anti-p38 MAPK, anti-Lef,anti-c-Myc and anti-Histone (Santa Cruz Biotechnology, Santa Cruz,Calif.), anti-GSK3β, anti-β-catenin, anti-phospho-p38 MAPK andantiphospho-S⁹ (Cell Signaling, Beverly, Mass.) and Anti-Flag(Stratagene, La Jolla, Calif.) were used for Western blot analysis. Thephospho-S³⁸⁹ GSK3β antibody was made by Proteintech, Chicago, Ill.,using N-C-ARIQAAA(phos-S)PPANATA (SEQ ID NO:87) for immunization.Anti-Akt, anti-p38 MAPK and Anti-GSK3β were used forimmunoprecipitations. Anti-rabbit-HRP, anti-mouse-HHP (JacksonImmunoResearch Laboratories, West Grove, Pa.) and anti-goat-HRP (SantaCruz Biotechnology, Santa Cruz, Calif.) were used as secondaryantibodies.

In vitro Kinase Assays

In vitro kinase assays for p38 MAPK and ERK were performed as describedin (28) with immunoprecipitated p38 MAPK, purified recombinant activep38 MAPK (Cell Signaling, Beverly, Mass.) and ERK (Cell Signaling,Beverly, Mass.) Kinase using 2 μg inactive wildtype GST-GSK3β, GST-GSK3βT⁴³A mutant, GST-GSK3β T³⁹⁰A mutant and GST-GSK3β T³⁹⁰A/T⁴³A doublemutant (2 βg), GST-GSK3α (2 μg) (Cell Signaling, Beverly, Mass.) (boiledto inactivate) and GST-ATF2 fusion protein (2 μg) (Cell Signaling,Beverly, Mass.) as substrates in the presence or absence of SB203580(2.5 μM) (Calbiochem, San Diego, Calif.). The reactions were terminatedafter 30 min at 30° C. by addition of SDS-PAGE sample buffer, separatedby SDS-PAGE and transferred to nitrocellulose. Protein was visualized bystaining with PonceauS and incorporated ³²P was visualized byautoradiography. In specific experiments, whole cell extracts were firstdepleted of Akt prior to p38 MAPK immunoprecipitation by beingpre-incubated with anti-Akt antibody prebound to protein A-Sepharosefollowed by one incubation with protein A-sepharose alone.

Active GST-GSK3β and GST-GSK3β-T⁴³A and GST-GSK3β-T³⁹⁰ mutants wereexpressed and purified as described in (29). The specific activity ofthe wildtype and the mutants was determined using the GSM substratepeptide (Upstate, Lake Placid, N.Y.) and were as follows: GST-GSK3β (91μmol/μg×min) and GST-GSK3β-T⁴³A (53 μmol/μg×min) and GST-GSK3β-T³⁹⁰A (65μmol/μg×min). In vitro GSK3β kinase assays were performed by incubatingpurified active wildtype GST-GSK3β or GST-GSK3β-T⁴³A and GST-GSK3β-T³⁹⁰Amutants (400 ng) at 30° C. for 10 minutes in kinase buffer supplementedwith 1 mM ATP containing [γy-³²P] ATP and GSM substrate peptide (62.5μM) (Upstate, Lake Placid, N.Y.). Reactions were terminated by spottingonto P81 filters. Filters were washed extensively and counted in ascintillation counter. To examine the effect of p38 MAPK and Akt onGSK3β activity, purified active GSK3β or mutants were preincubated for15 minutes at 30° C. with 500 ng recombinant active p38 MAPK or activeAkt (Cell Signaling, Beverly, Mass.) prior to performing the GSK3βactivity assay as described above.

For peptide inhibition assays, Phospho-S⁹ (GHPRTTS(PO₃H₂)FAE) (SEQ IDNO:95) (20). Phospho-T³⁸⁰ (RIQAAAST(PO₃H₂)PTN) (SEQ ID NO:7) andnon-phospho-T³⁹⁰ (RIQAAASTPTN) (SEQ ID NO:96) peptides were synthesizedand purified by UVM protein Core Facility. In vitro kinase assays wereperformed with purified GST-GSK3β (400 ng) in Kinase buffer supplementwith 1 mM ATP containing [γ-³²P] ATP and the indicated concentrations ofGSM and inhibitory peptide.

Mass Spectrometric Analysis by LC-MS/MS

Kinase-inactive GSK3β was preincubated with or without activerecombinant p38 MAPK for 30 min in the presence of ATP. The reaction mixwas treated with an ammonium bicarbonate buffer containing DTT (10 mM)to reduce cysteines, incubated with iodoacetamide and digested withtrypsin (40 ng/μl). Digestion was stopped with acetic acid (10%),centrifuged and the supernatant was used for analysis by electrosprayionization (ESI) liquid chromatography-mass spectrometry (LC-MS). Afused-silica microcapillary LC column (12 cm×75 μm id) packed with C18reversed phase resin (Magic C18AQ, 5-μm particle size, 20-nm pore sise,Michrom Bioresources, Auburn, Calif.) was used with nanospray ESI. Thenanospray-ESI was fitted onto a linear quadrupole ion trap (LTQ) massspectrometer (Thermo Electron, San Jose, Calif.) that was operated in acollisional-induced dissociation mode to obtain both MS and MS/MSspectra. Samples of tryptic peptides were loaded onto the microcapillarycolumn and separated by applying a gradient from 5-80% acetonitrile in0.5% acetic acid at a flow rate of 250 nL/min for 55 min. Massspectrometry data were acquired in data-dependent acquisition mode, inwhich a full MS scan was followed by 10 MS/MS spectra of the 10 mostabundant ions. Spectra were searched against the human InternationalProtein Index (IPI) database using SEQUEST (Bioworks software package,version 3.3, Thermo Electron, San Jose, Calif.). GSK3β (Swiss-Prot entryP49841) was identified. The search parameters permitted ±1.0 Da peptideMS tolerance, and ±1.0 Da MS/MS tolerance. Phosphorylation (a +80 massincrease) was sought on serine (S), threonine (T), and tyrosine (Y)residues, together with allowance of oxidation of methionine andcarboxymethylatlon of cysteines. Up to two missed tryptic cleavages ofpeptides were considered. The cutoff for SEQUEST assignment was across-correlation score (Xcorr) greater than 1,9, 2.5, and 3.8 forpeptide charge states of 1,2, and 3, respectively; and adelta-correlation score (ΔCn) >0.10. Manual identifications were alsoperformed to define the exact location of phosphorylation sites basedupon the b- and y-ions of the corresponding identifying MS/MS spectra.

Results and Discussion

The increased abundance of c-Myc and Lef proteins in the MKK6 transgenicthymocytes compared with Rag^(−/−) thymocytes was confirmed by Westernblot analysis (FIG. 1A). Thymocytes from Rag^(−/−) crossed with MKK6transgenic (Rag^(−/−) MKK6) mice contained higher amounts of c-Myc andLef proteins than did Rag^(−/−) thymocytes, indicating that theactivation of p38 MAPK, but not the pre-TCR signals, contribute to theenhanced expression of these transcription factors (FIG. 1B). The c-mycand lef genes are targets of the β-catenin signaling pathway in certaincontests (12, 13). Nuclear accumulation of β-catenin was detected inMKK6 thymocytes, but not in Rag^(−/−) thymocytes (FIG. 1C). Expressionof constitutively active MKK6 in 293T cells was also sufficient toincrease the amount of β-catenin protein (FIG. 1D), but had no effect onβ-catenin mRNA (FIG. 6).

Phosphorylation of β-catenin by GSK3β targets β-catenin forubiquitination and subsequent degradation (14, 15). The bestcharacterised mechanism for the inactivation of GSK3β is throughphosphorylation of its N-terminus at Ser⁹ by Akt (16). No increase wasobserved in the amount of phospho-Ser⁹ GSK3β in MKK6 thymocytes comparedwith that in Rag^(−/−) thymocytes (FIG. 2A). Similarly, no increase inphospho-Ser⁹ was observed in 293T cells transfected with constitutivelyactive MKK6 (FIG. 28). Phosphorylation of Ser⁹ was impaired byWortmanin, an inhibitor of the PI3K-Akt pathway, but it was not affectedby the pharmacological inhibitor of p38 MAPK SB203580 (FIG. 7). Thus,p38 MAPK appears not to regulate the Akt-mediated phosphorylation ofGSK3β on Ser⁹. p38 MAPK immunoprecipitated from MKK6 thymocytes orMKK6-transfected 293T cells phosphorylated recombinantcatalytically-inactive GSK3β in vitro and this phosphorylation wasblocked by the p38 MAPK inhibitor (FIG. 2C). No Akt was detected in p38MAPK immunoprecipitates and no p38 MAPK was detected in Aktimmunoprecipitates (FIG. 8) ruling out the presence of residual AKTassociated with p38 MAPK. Depletion of Akt before immunoprecipitatingp38 MAPK, did not affect phosphorylation of GSK3β (FIG. 2D). A purifiedrecombinant activated p38 MAPK also phosphorylated GSK3β in vitro andthis phosphorylation was blocked by SB203580 (FIG. 2E).Co-immunoprecipitation analysis showed that p38 MAPK was present inGSK3β immunoprecipitates from MKK6 thymocytes (FIG. 2F) and 293T cells(FIG. 9). Thus, p38 MAPK physically associates with and phosphorylatesGSK3β at a Ser⁹-independent residue. Although GSK3α and GSK3β arethought to be similarly regulated and can compensate for each other forsome functions (17, 18), GSK3α was not phosphorylated by recombinant p38MAPK in vitro (FIG. 2G).

The MAPK extracellular signal regulated protein kinase (ERK)phosphorylates Thr⁴³ of GSK3β(19), but does not affect GSK3β activity.Although SerPro or ThrPro motifs recognized by ERK are also recognizedby other MAPK groups, p38 MAPK was still able to partially phosphorylatea GSK3β-T⁴³A mutant (FIG. 3A), suggesting the existence of additionalphosphorylation sites in GSK3β. Mass spectrometry analysis ofrecombinant GSK3β phosphorylated in vitro by p38 MAPK showed two GSK3βphosphopeptides containing phosphorylation within a consensus SerPro orThrPro motif, a phospho-peptide containing Thr⁴³ and a C-terminalpeptide (384-403) containing the Thr³⁹⁰Pro motif (corresponding toSer³⁸⁹Pro in mouse GSK3β) (FIGS. 10 and 11). To confirm Thr³⁹⁰ as atarget of p38 MAPK in GSK3β, catalytically-inactive GSK3β-T³⁹⁰A andGSK3β-T⁴³A/T³⁹⁰A mutants were used as substrates for p38 MAPK in vitro.Phosphorylation of the GSK3β-T³⁹⁰A mutant by p38 MAPK was partiallyreduced but not abrogated (FIG. 3B), but phosphorylation of theGSK3β-T⁴³A/T³⁹⁰A mutant was abrogated, indicating that these tworesidues are likely the targets for p38 MAPK in GSK3β. The T⁴³A mutationbut not the T³⁹⁰A mutation abrogated phosphorylation of GSK3β by ERK(FIG. 3C). Thus, Thr³⁹⁰ of GSK3β appears to be specificallyphosphorylated by p38 MAPK.

The activity of wild-type GSK3β and GSK3β-T⁴³A and GSK3β-T³⁹⁰A mutants,before or after incubation with p38 MaPK or Akt was examined. p38 MAPKinhibited both wild-type GSK3β and GSK3β-T⁴³A mutant (FIG. 3D-E), butnot the GSK3β-T³⁹⁰A mutant (FIG. 3F). Akt inhibited wild-type GSK3β andthe two mutants (FIG. 3D-F). p38 MAPK did not affect the activity ofGSK3α (FIG. 12) in which the Thr³⁹⁰ residue from GSK3β is not conserved.Together, these results demonstrate that p38 MAPK-mediatedphosphorylation of GSK3β at Thr³⁹⁰ (but not Thr⁴³) is sufficient toinhibit GSK3β activity. A peptide derived from the N-terminus of GSK3βcontaining phospho-Ser⁹ specifically inhibits GSK3β in vitro (20, 21). Aphospho-Thr³⁹⁰ peptide also inhibited GSK3β activity, while theunphosphorylated-Thr³⁹⁰ peptide did not (FIG. 3G). The phospho-Thr³⁹⁰peptide inhibited GSK3β activity as efficiently as the phospho-Ser⁹peptide (FIG. 3H-J). Thus, phosphorylation at Thr³⁹⁰ by p38 MAPK maycause an inhibition of GSK3β comparable to the phosphorylation of Ser⁹by Akt.

To demonstrate the phosphorylation of this residue in intact cells andin vivo, an antibody (Ab) specific to a mouse phospho-Ser³⁸⁹ GSK3βpeptide was generated. A band corresponding to GSK3β was detected withthis Ab in wild-type and GSK3α^(−/−) embryonic stem (ES) cells, but notin the GSK3β^(−/−)ES cells by Western blot analysis (FIG. 4A). Thisspecific band was also present in GSK3β^(−/−) ES cells transacted with awild-type GSK3β, but not with a GSK3β-S³⁸⁹A mutant (FIG. 4B).Phospho-Ser³⁸⁹ GSK3β was detected in mouse GSK3β-transfected 293T cells,but only if active MKK6 was present (FIG. 4C). The presence of thephospho-Ser³⁸⁹ GSK3β in these cells correlated with an increased amountof β-catenin (FIG. 4C), indicative of an inhibition of GSK3β activity.Ser³⁸⁹-phosphorylation was also detected in wild-type GSK3β, but not theGSK3β-S³⁸⁹A mutant after in vitro incubation with activated p38 MAPK(FIG. 13). Phosphatase treatment of GSK3β previously incubated withactivated p38 MAPK abrogated its recognition by the phospho-Ser³⁸⁹ Ab(FIG. 13). Together, these results show the specificity of this Ab forphospho-S³⁸⁹ GSK3β, and the phosphorylation of GSK3β as S³⁸⁹ by p38 MAPKin vitro.

To determine whether activation of p38 MAPK was required forphosphorylation of GSK3β at Ser³⁸⁹ in intact cells mouseGSKβ-transfected 293T cells were treated with SB203580. Inhibition ofp38 MAPK abrogated the phosphorylation of Ser³⁸⁹ (FIG. 4D). Similarly,treatment with SB203580 inhibited phosphorylation of endogenous GSK3β atSer³⁸⁹ in wild-type mouse embryonic fibroblasts (MEFs) and embryonicstem (ES) cells (FIG. 4E-F). The abundance of phospho-Ser³⁸⁹ in MEFsdeficient for the major upstream activators of p38 MAPK, MKK3 and MKK6was also examined (22). Phospho-Ser³⁸⁹ was barely detectable inMKK3^(−/−)MKK6^(−/−)MEFs (FIG. 4G). In contrast, the amounts ofphospho-Ser⁹ were comparable in wildtype and MKK3^(−/−)MKK6^(−/−)MEFs(FIG. 4G). Thus, activation of p38 MAPK appears to be required forphosphorylation of GSK3β at Ser³⁸⁹. Inhibition of p38 MAPK either bySB203580 (FIG. 4D) or by the absence of MKK3 and MKK6 (FIG. 4I) alsodecreased the amount of β-catenin, consistent with the possibility thatp38 MAPK activation is required for repressing GSK3β activity.

Phospho-Ser³⁸⁹ was also examined in different mouse tissues. A highamount of phospho-S³⁸⁹ was detected in brain and lesser amounts wheredetected in thymocytes and spleen cells (FIG. 4J-K). Phospho-Ser³⁸⁹ wasnot detected in kidney (FIG. 4J-K), liver or heart (FIG. 14).Phosphorylation of GSK3β at Ser⁹ was detected in practically allexamined tissues (FIG. 4J-K). Analysis of the relative abundance ofphospho-S³⁸⁹ and phospho-S⁹ showed a predominance of the former in brainand thymocytes (FIG. 4J-K), which correlated with the selective highactivation of p38 MAPK in these tissues (FIG. 15). Inhibition of p38MAPK by treating animals with SB203580 reduced the levels ofphospho-Ser³⁸⁹ GSK3β in both thymocytes and brain (FIG. 4L). Analysis ofphospho-Ser³⁸⁹ in MKK6 and Rag^(−/−) thymocytes showed phospho-S³⁸⁹GSK3β was present selectively in MKK6 thymocytes (FIG. 4M). Together,there results support the proposal that GSK3β is phosphorylated at S³⁸⁹in vivo by p38 MAPK and that this alternative regulatory mechanism ofGSK3β is tissue specific.

To date phosphorylation at Ser⁹ by Akt is the best characterisedmechanism for the inhibition of GSK3β activity. However, knockin micewhere Ser⁹ was replaced by Ala have only a subtle defect related toinsulin regulation of glycogen synthase in skeletal muscle (23),indicating that alternative mechanisms may be involved in the negativeregulation of GSK3β for certain functions. Herein it is proposed thatphosphorylation of GSK3β at S³⁸⁹ by p38 MAPK may be one such mechanism.Conditions that promote the activation of p38 MAPK promote theaccumulation of β-catenin in certain scenarios, thus activation of thep38 MAPK pathway could be an alternative mechanism to regulateβ-catenin/TCF signaling (and potentially, cell survival) throughinactivation of GSK3β.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. All references,including patent documents, disclosed herein are incorporated byreference in their entirety.

1. A method of reducing activity of GSK3β in a cell, the methodcomprising: contacting the cell with one or both of (a) a compositioncomprising an isolated GSK3β polypeptide, wherein the polypeptide is afull-length GSK3β protein or a fragment of a full-length GSK3β proteinand comprises a phosphorylated residue that corresponds to residueThr³⁹⁰ in a full-length, wild-type, human GSK3β amino acid sequence, orcorresponds to residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3βamino acid sequence, and (b) a composition comprising a compound thatresults in the phosphorylation of a GSK3β residue that corresponds toresidue Thr³⁹⁰ in a full-length, wild-type, human GSK3β amino acidsequence, or corresponds to the residue Ser³⁸⁹ in a full-length,wild-type, mouse GSK3β amino acid sequence.
 2. The method of claim 1,wherein the GSK3β polypeptide is a human GSK3β polypeptide and thephosphorylated residue corresponds to the Thr³⁹⁰ residue.
 3. The methodof claim 2, wherein the fragment comprises the amino acid sequence setforth as: RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7).
 4. The method of claim 1,wherein, the GSK3β polypeptide is a mouse GSK3β polypeptide and thephosphorylated residue corresponds to the Ser³⁸⁹ residue.
 5. The methodof claim 4, wherein the fragment comprises the amino acid sequence setforth as ARIQAAAS(PO₃H₂)PPANATA (SEQ ID NO:87).
 6. The method of claim1, wherein the compound that results in the phosphorylation of the GSK3βresidue modulates expression or activity of a component of the p38 MAPKsignaling pathway in the cell
 7. The method of claim 6, wherein thecompound that results in the phosphorylation of the GSK3β residueincreases expression or activity of p38 MAPK.
 8. The method of claim 7,wherein the compound that results in the phosphorylation of the GSK3βresidue comprises p38 MAPK.
 9. A method for treating a disease orcondition associated with GSK3β activity, method comprising:administering to a subject having a disease or condition associated withGSK3β activity one or both of (a) a therapeutically effective amount ofa composition comprising a GSK3β polypeptide, wherein the polypeptide isa full-length GSK3β protein or a fragment of a full-length GSK3β proteinand comprises a phosphorylated residue that corresponds to residueThr³⁹⁰ in a full-length, wild-type, human GSK3β amino acid sequence, orcorresponds to residue Ser³⁸⁹ in a full-length, wild-type, mouse GSK3βamino acid sequence and (b) a therapeutically effective amount of acomposition comprising a compound that results in the phosphorylation ofa GSK3β residue that corresponds to residue Thr³⁹⁰ in a full-length,wild-type, human GSK3β amino acid sequence, or corresponds to residueSer³⁸⁹ in a full-length, wild-type, mouse GSK3β amino acid sequence. 10.The method of claim 9, wherein the GSK3β polypeptide is a human GSK3βpolypeptide and the phosphorylated residue corresponds to the Thr³⁹⁰residue.
 11. The method of claim 10, wherein the polypeptide comprisesthe amino acid sequence set forth as: RIQAAAST(PO₃H₂)PTN (SEQ ID NO:7).12. The method of claim 9, wherein the GSK3β polypeptide is a mouseGSK3β polypeptide and the phosphorylated residue corresponds to theSer³⁸⁹ residue.
 13. The method of claim 12, wherein the fragmentcomprises the amino acid sequence set forth as ARIQAAA S(PO₃H₂)PPANATA(SEQ ID NO:87).
 14. The method of claim 9, wherein the compound thatresults in the phosphorylation of the GSK3β residue modulates expressionor activity of a component of the p38 MAPK signaling pathway in thecell.
 15. The method of claim 9, wherein the compound that results inthe phosphorylation of the GSK3β residue increases expression oractivity of p38 MAPK.
 16. The method of claim 15, wherein the compoundthat results in the phosphorylation of the GSK3β residue comprises p38MAPK.
 17. The method of claim 9, wherein the disease or condition is aneurological disease or condition, and is optionally Alzheimer's diseaseor bipolar disorder.
 18. A method of increasing activity of GSK3β in acell, the method comprising: contacting the cell with a compositioncomprising a compound that reduces phosphorylation of a residue thatcorresponds to residue Thr³⁹⁰ in a full-length, wild-type, human GSK3βamino acid sequence, or corresponds to residue Ser³⁸⁹ in a full-length,wild-type, mouse GSK3β amino acid sequence.
 19. The method of claim 18,wherein the compound comprises a compound that modulates expression oractivity of a component of the p38 MAPK signaling pathway in the cell.20. A method of identifying a compound that modulates GSK3β activity,the method comprising: (a) contacting a GSK3β polypeptide of or fragmentthereof comprising a phosphorylated residue that corresponds to residueThr³⁹⁰ in a full-length, wild-type, human GSK3β amino acid sequence,with p38 MAPK and a putative modulating compound under suitableconditions for phosphorylation of the residue that corresponds toresidue Thr³⁹⁰ in a full-length, wild-type, human GSK3β amino acidsequence; (b) detecting the level of phosphorylation of the GSK3βresidue that corresponds to residue Thr³⁹⁰ in a full-length, wild-type,human GSK3β amino acid sequence, in the contacted polypeptide; and (c)comparing the level of phosphorylation of the GSK3β residue thatcorresponds to residue Thr³⁹⁰ in a full-length, wild-type, human GSK3βamino acid sequence, in the contacted polypeptide to a level ofphosphorylation of the corresponding residue in a control GSK3βpolypeptide not contacted with the compound, wherein if the level ofphosphorylation is higher in the contacted polypeptide than in thecontrol polypeptide the compound is identified as an inhibitor of GSK3βactivity and if the level of phosphorylation is lower in the contactedpolypeptide than in the control polypeptide the compound is identifiedas an enhancer of GSK3β activity.